Efficacy of a novel polylactic acid/nanoclay (NK-75) biodegradable composite scaffold in healing critical-size bone defects: an experimental study in a rabbit ulnar model

Efficacy of a novel polylactic acid/nanoclay (NK-75) biodegradable composite scaffold in healing critical-size bone defects: an experimental study in a rabbit ulnar model | Research Square window.SnipcartSettings = { analytics: { enabled: false } }; (function() { var accessVector = localStorage.getItem('access_vector') || ''; window.dataLayer = window.dataLayer || []; if (accessVector) { window.dataLayer.push({ user: { profile: { profileInfo: { snid: accessVector } } } }); } })(); (function(w,d,s,l,i){w[l]=w[l]||[];w[l].push({'gtm.start':new Date().getTime(),event:'gtm.js'});var f=d.getElementsByTagName(s)[0],j=d.createElement(s),dl=l!='dataLayer'?'&l='+l:'';j.async=true;j.src='https://www.googletagmanager.com/gtm.js?id='+i+dl;f.parentNode.insertBefore(j,f);})(window,document,'script','dataLayer','GTM-K279D39R'); Browse Preprints In Review Journals COVID-19 Preprints AJE Video Bytes Research Tools Research Promotion AJE Professional Editing AJE Rubriq About Preprint Platform In Review Editorial Policies Our Team Advisory Board Help Center Sign In Submit a Preprint Cite Share Download PDF Research Article Efficacy of a novel polylactic acid/nanoclay (NK-75) biodegradable composite scaffold in healing critical-size bone defects: an experimental study in a rabbit ulnar model Ashish Ranjan, Ajit Singh, Pralay Maiti, Sanjay Yadav, Amrita Ghosh, and 4 more This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-8882301/v1 This work is licensed under a CC BY 4.0 License Status: Under Review Version 1 posted 8 You are reading this latest preprint version Abstract Background Critical-size bone defects remain a formidable challenge in orthopaedic surgery. Biodegradable polymer scaffolds represent a promising alternative to autologous bone grafting, but conventional polylactic acid (PLA) scaffolds exhibit limited osteoconductivity and generate acidic degradation products unfavorable to bone healing. Incorporation of nanoclay particles into PLA matrices may overcome these limitations. This study evaluated a novel PLA/nanoclay (NK-75) biodegradable composite scaffold for healing critical-size bone defects in a rabbit ulnar model. Methods New Zealand White rabbits underwent bilateral mid-diaphyseal ulnar osteotomy creating 3 mm extraperiosteal critical-size defects. Defects were assigned to three groups: PLA + NK-75 composite scaffold (n = 15 observations), PLA-only scaffold (n = 4), or untreated empty defect control (n = 11). Animals were sequentially sacrificed at 4, 8, 12, 16, and 20 weeks post-surgery. Radiological healing was assessed using the Lane-Sandhu scoring system (total score 0–10, comprising bone formation, proximal union, distal union, and remodeling). Data were analyzed using Kruskal-Wallis test with Dunn's post-hoc comparisons, Mann-Whitney U test, linear mixed models, generalized estimating equations, ordinal logistic regression, and permutation tests. Effect sizes and post-hoc power were calculated. Results The PLA + NK-75 group demonstrated significantly higher mean total Lane-Sandhu scores (7.20 ± 1.93) compared to PLA (4.75 ± 3.59) and control (4.00 ± 2.32; Kruskal-Wallis p = 0.0144). Post-hoc analysis confirmed PLA + NK-75 superiority over control (Dunn's p = 0.0062; Mann-Whitney p = 0.0044). Large effect sizes were observed (Hedges' g = 1.47; Cliff's delta = 0.66). The probability that a randomly selected PLA + NK-75 observation exceeds a control observation was 83.0%. Excellent healing (score ≥ 8) was achieved in 46.7% of PLA + NK-75 observations versus 0% in controls (Fisher's exact p = 0.0025). PLA + NK-75 was the only group demonstrating remodeling activity. A significant ordered trend (CONTROL < PLA < PLA + NK-75) was confirmed by the Jonckheere-Terpstra test (p = 0.0021). Results were consistent across all parametric, non-parametric, and permutation-based analyses. Achieved statistical power was 85.5%. Conclusion The PLA/NK-75 biodegradable composite scaffold significantly enhances radiological bone healing with large effect sizes and accelerated milestone achievement. These findings support nanoclay-enhanced biodegradable scaffolds as a promising strategy for orthopaedic bone regeneration. Bone regeneration Critical-size bone defect Polylactic acid Nanoclay Biodegradable scaffold NK-75 Lane–Sandhu score Rabbit ulnar model Bone tissue engineering Nanocomposite Figures Figure 1 Figure 2 Figure 3 Figure 4 Figure 5 Figure 6 INTRODUCTION Critical-size bone defects arising from trauma, tumour resection, infection debridement, and revision surgery remain among the most challenging problems in contemporary orthopaedic practice [1,2]. Fracture nonunion complicates an estimated 5–10% of all fractures and imposes a substantial clinical and socioeconomic burden through prolonged disability, repeated surgical interventions, and diminished quality of life [1]. Autologous bone grafting continues to be regarded as the gold standard owing to its combined osteogenic, osteoinductive, and osteoconductive properties; however, its utility is constrained by limited graft volume, donor-site morbidity, and the requirement for a second operative site [2,3]. These limitations have stimulated considerable research into synthetic scaffold-based alternatives that can be manufactured reproducibly and made available off the shelf [3]. Biodegradable polymer scaffolds have emerged as a promising bone tissue engineering strategy, providing temporary mechanical support while gradually resorbing and being replaced by regenerating bone [4,5]. Among the biodegradable polymers, polylactic acid (PLA) has attracted particular attention because of its established biocompatibility, FDA approval for clinical use, tunable degradation kinetics, and favourable mechanical properties [6,7]. Nevertheless, PLA exhibits several recognised shortcomings, including limited osteoconductivity, a relatively hydrophobic surface that impedes cellular adhesion, and the generation of acidic degradation products that can provoke local inflammation and impair osteoblast function [6]. To address these limitations, the incorporation of nanoscale inorganic fillers into polymer matrices has been explored extensively [8]. Nanoclays, particularly montmorillonite-based layered aluminosilicates, represent an attractive class of bioactive nanofillers owing to their high surface area, cation exchange capacity, and established safety in biomedical applications [9]. Recent investigations have demonstrated that nanoclay-functionalised scaffolds significantly enhance osteogenic differentiation of mesenchymal stem cells in vitro and promote bone regeneration in vivo through multiple mechanisms, including mechanical reinforcement, sustained release of bioactive ions (silicon, magnesium), buffering of acidic degradation byproducts, and improved surface hydrophilicity for cell attachment [10–12]. Furthermore, polymer-nanoclay nanocomposites have shown promise in bone cement and drug delivery systems [13,14]. Despite these encouraging findings, in vivo evaluation of PLA/nanoclay composites specifically employing organically modified montmorillonite (NK-75) in an orthopaedic bone defect model has not been reported. The present study aimed to evaluate the efficacy of a novel PLA/NK-75 biodegradable composite scaffold in healing critical-size bone defects compared to PLA alone and untreated empty defect controls in a rabbit ulnar model, with the hypothesis that nanoclay augmentation would produce superior radiological healing in an ordered dose-response pattern (CONTROL < PLA < PLA + NK-75). MATERIALS AND METHODS Ethical Approval This study was approved by the Institutional Animal Ethics Committee (IAEC), Institute of Medical Sciences, Banaras Hindu University, Varanasi (Registration No. 542/GO/ReBi/S/02/CPCSEA, dated 26.05.2017), and conducted in accordance with the guidelines of the Committee for the Purpose of Control and Supervision of Experiments on Animals (CPCSEA), Government of India. The study is reported following the ARRIVE 2.0 guidelines for animal research. Scaffold Preparation Materials Polylactic acid (PLA) served as the biodegradable polymer matrix. NK-75 nanoclay, an organically modified montmorillonite (ion-exchanged with dimethyl dihydrogenated tallow ammonium; cation exchange capacity 70 mequiv. per 100 g), was selected as the bioactive nanofiller. Elemental composition of NK-75 was confirmed using energy-dispersive X-ray analysis (EDAX). Scaffold preparation was performed in collaboration with the School of Material Sciences and Technology, IIT-BHU. Composite Fabrication PLA+NK-75 nanohybrid composites were prepared by solution-mixing PLA with 5 wt% NK-75 nanoclay using a handheld mixer for 10 minutes to achieve uniform nanoclay dispersion. The composite was molded into cylindrical scaffolds of 3 mm length matching the planned defect size. PLA-only scaffolds were prepared using an identical protocol without nanoclay addition. All scaffolds were sterilized prior to surgical implantation. Animal Model and Experimental Design Animals Skeletally mature New Zealand White rabbits ( Oryctolagus cuniculus ), aged 6 months and weighing up to 3 kg, of both sexes, were obtained from the institutional animal facility, IMS-BHU. Animals were housed under standard conditions with ad libitum access to food and water, 12-hour light-dark cycles, and were acclimatized for one week prior to surgery. Study Groups and Sample Size The study comprised 30 experimental observations distributed across three groups. The PLA+NK-75 group (n=15 observations) served as the primary experimental arm. The PLA-only group (n=4 observations) served as the polymer control to isolate the contribution of nanoclay. The untreated empty defect group (n=11 observations) served as the negative control. Animals underwent bilateral ulnar osteotomy, with each limb serving as an independent observation. Animals were sequentially sacrificed at five time points (weeks 4, 8, 12, 16, and 20), with the distribution reflecting the primary focus on early-to-mid healing phases: week 4 (n=9), week 8 (n=8), week 12 (n=6), week 16 (n=5), and week 20 (n=2). Unequal group sizes were accounted for using non-parametric statistical methods and confirmed with post-hoc power analysis. Surgical Procedure Rabbits were anesthetized with an intramuscular gluteal injection of ketamine (30 mg/kg) and midazolam (1 mg/kg). Bilateral forearms were shaved and disinfected with povidone-iodine. Through a direct approach to the mid-diaphysis of the ulna, a 3 mm segmental osteotomy was performed extraperiosteally, creating a critical-size defect. In treatment groups, a 3 mm scaffold (PLA+NK-75 or PLA-only) was press-fit into the defect gap. Control limbs received no scaffold, leaving the defect empty. Wound closure was performed in layers using absorbable (Vicryl) and non-absorbable (Nylon/Prolene) sutures. Three postoperative doses of gentamicin (10 mg) were administered for infection prophylaxis. Animals were allowed unrestricted cage activity postoperatively. Sacrifice and Specimen Harvest At each designated time point, rabbits were humanely euthanized by intravenous injection of sodium pentobarbital (>100 mg/kg). Bilateral ulnar specimens were harvested en bloc with surrounding soft tissue for radiological assessment. Outcome Assessment Radiological Scoring — Lane-Sandhu System Anteroposterior radiographs of harvested specimens were obtained at each time point . Radiological healing was assessed using the Lane-Sandhu scoring system, a validated semi-quantitative grading scale comprising four components: bone formation (0–4 points: 0=no new bone, 1=25% of defect filled, 2=50%, 3=75%, 4=100%), proximal union (0–2 points: 0=no union, 1=partial, 2=complete), distal union (0–2 points: same criteria), and remodeling (0–2 points: 0=none, 1=recanalization of medullary canal, 2=cortical remodeling). The total score ranged from 0 (no healing) to 10 (complete healing with remodeling). Total scores were further categorized as: No Healing (0), Poor (1–3), Fair (4–5), Good (6–7), and Excellent (8–10). Histological Assessment Following radiological assessment, specimens were processed for histological evaluation. Sections from the implant site and adjacent tissues were obtained for gross examination (bone status, soft tissue status, wound integrity) and microscopic evaluation using hematoxylin and eosin (H&E) for general cellular architecture, Toluidine Blue O for demonstration of mineralized bone, osteoid seams, and osteoblast/osteoclast identification, and Von Kossa staining for mineralization assessment. Osteoblastic activity and periscaffold tissue response were evaluated qualitatively. Statistical Analysis All analyses were performed using IBM SPSS Statistics version 29.0 (IBM Corp., Armonk, NY, USA). Descriptive statistics are reported as mean ± standard deviation (SD), median, interquartile range (IQR), and range. The primary analysis employed the Kruskal-Wallis test for overall group comparison, followed by Dunn's post-hoc test with Bonferroni correction and Mann-Whitney U tests for pairwise comparisons. Fisher's Exact Test assessed the association between treatment group and categorical healing outcomes. The Jonckheere-Terpstra test evaluated the a priori ordered alternative hypothesis (CONTROL < PLA < PLA+NK-75). Spearman's rank correlation assessed the relationship between time and healing scores. Sensitivity analyses included a linear mixed model with animal ID as a random effect, generalized estimating equations (GEE) with exchangeable correlation structure, ordinal logistic regression (cumulative link model) for ordered healing categories, and Poisson regression for count-modeled total scores. Negative binomial regression was performed to assess overdispersion. Robust regression using M-estimation was performed to evaluate outlier influence. Effect sizes were calculated using Cliff's delta (non-parametric), Hedges' g and Glass's Δ (standardized mean differences), epsilon-squared and eta-squared (variance explained), and Cohen's f. The common language effect size (probability of superiority) was computed for each pairwise comparison. Bootstrap confidence intervals for group medians were generated using 10,000 resampling iterations. Permutation testing (10,000 iterations) provided exact p-values free from distributional assumptions. Internal consistency of the Lane-Sandhu scale was evaluated using Cronbach's alpha. Principal component analysis (PCA) assessed the dimensional structure of the four scoring components. Post-hoc power analysis determined achieved statistical power. Model comparison using Akaike Information Criterion (AIC) and Bayesian Information Criterion (BIC) identified the optimal predictor combination. A two-sided p-value < 0.05 was considered statistically significant. RESULTS Sample Characteristics and Overall Healing Scores Thirty observations across three groups and five time points were analyzed (Figure 1,2 and 3). No perioperative mortality, wound infection, or scaffold-related adverse events were recorded. The descriptive statistics and component-level Lane-Sandhu scores are presented in Table 1 . The PLA+NK-75 group demonstrated the highest mean total score of 7.20 ± 1.93, exceeding both PLA (4.75 ± 3.59) and control (4.00 ± 2.32). The PLA+NK-75 group was the only group to achieve the maximum possible score of 10, while the control group's maximum was limited to 7. Across all four Lane-Sandhu components, PLA+NK-75 showed consistent superiority. Bone formation was highest in PLA+NK-75 (3.47 ± 0.64) compared to control (2.27 ± 1.01) and PLA (2.25 ± 1.71). Distal union showed the most pronounced intergroup differences (PLA+NK-75: 1.47 ± 0.52 vs. control: 0.55 ± 0.52). Notably, remodeling activity was exclusively observed in the PLA+NK-75 group (0.47 ± 0.74), with no remodeling in either control or PLA groups throughout the entire observation period, indicating that only the nanoclay-enhanced composite achieved sufficient healing maturity to initiate the final phase of bone regeneration. Temporal Healing Progression and Categorical Outcomes A clear temporal progression was observed, with overall mean scores increasing from 3.22 ± 2.44 at week 4 to 8.50 ± 2.12 at week 20 (Spearman ρ = 0.705, p < 0.0001). Group-wise temporal trajectories, illustrated in Figure 4 , revealed striking differences. PLA+NK-75 demonstrated superior scores at every time point — 5.2 at week 4 rising to 10.0 by week 20 — whereas controls progressed from 1.0 to only 7.0 over the same period. PLA+NK-75 achieved a score of 9.0 by week 12, a level the control group never attained even by week 20. Comparative serial radiographs across all three groups at 0 days, 8 weeks, and 16 weeks further illustrated the superior healing trajectory in PLA+NK-75 relative to PLA and control groups (Figure 5). Time-to-milestone analysis further highlighted the accelerated healing in PLA+NK-75. Good healing (score ≥6) was first achieved at week 4 in PLA+NK-75, compared to week 8 in controls and week 12 in PLA. Excellent healing (score ≥8) was attained by 7 PLA+NK-75 observations beginning at week 8, while controls never reached this threshold. The Friedman test confirmed significant within-group time progression only in PLA+NK-75 (χ² = 11.92, p = 0.018), indicating sustained progressive healing without plateau — a clinically desirable property for bone regeneration scaffolds. Categorical healing outcomes demonstrated marked intergroup differences. Excellent healing (score 8–10) was achieved in 46.7% (7/15) of PLA+NK-75 observations, compared to 25.0% (1/4) in PLA and 0% (0/11) in controls. Conversely, poor healing (score 1–3) was predominantly observed in controls (36.4%, 4/11), with only a single instance in PLA+NK-75 (6.7%). Fisher's Exact Test confirmed a significant association between treatment group and healing category (p = 0.0025). Histological evaluation corroborated the radiological findings. Comparative histological sections across groups and time points further demonstrated the superior tissue quality and progressive maturation in the PLA+NK-75 group relative to PLA and control groups (Figure 6). Statistical Analyses, Effect Sizes, and Model Validation The complete results of all statistical tests are summarized in Table 2 . The Kruskal-Wallis test demonstrated significant overall group differences for total score (χ² = 8.48, p = 0.0144), bone formation (χ² = 8.99, p = 0.0112), and distal union (χ² = 10.73, p = 0.0047). Proximal union did not reach significance (p = 0.1067), while remodeling approached significance (p = 0.0563). Dunn's post-hoc analysis with Bonferroni correction identified PLA+NK-75 as significantly superior to control (Z = −2.870, p = 0.0062), while PLA did not differ significantly from control (p = 0.7531). Mann-Whitney U testing corroborated these findings (PLA+NK-75 vs. control: U = 28.0, p = 0.0044). The Jonckheere-Terpstra test confirmed a significant ordered trend in the hypothesized direction — CONTROL < PLA < PLA+NK-75 (JT = 207, p = 0.0021) — validating the biological rationale that nanoclay augmentation of PLA provides additive benefit. Sensitivity analyses uniformly confirmed the primary findings. The linear mixed model, accounting for repeated measures within animals, estimated a PLA+NK-75 effect of +3.58 points over control (95% CI: 2.55–4.61, t = 6.82) after adjusting for time. Each additional week contributed 0.385 points (95% CI: 0.31–0.47, t = 9.48). GEE analysis with exchangeable correlation structure yielded consistent results (PLA+NK-75: Wald χ² = 64.02, p < 0.001). Ordinal logistic regression demonstrated dramatically increased odds of achieving higher healing categories in PLA+NK-75 (OR = 673.24, p < 0.001), while Poisson regression yielded a rate ratio of 1.91 (95% CI: 1.35–2.74, p < 0.001), indicating 91% higher expected healing scores. Permutation testing with 10,000 iterations produced an exact p-value of 0.008, more stringent than the asymptotic Kruskal-Wallis p-value. Across all nine analytical methods, PLA+NK-75 versus control remained significant at p < 0.01, demonstrating exceptional robustness. Effect size measures and power analysis are presented in Table 3 . All metrics consistently indicated large treatment effects. Cliff's delta of 0.661 signifies that in 66.1% of all possible pairings, a PLA+NK-75 observation exceeds a control observation. Hedges' g of 1.472 and Glass's Δ of 1.377 both substantially exceed the conventional threshold of 0.8 for large effects. At the global level, eta-squared of 0.323 indicates that group membership explains 32.3% of total variance in healing scores. The common language effect size translates to an 83.0% probability of PLA+NK-75 superiority over control — approximately 5:1 odds. Post-hoc power analysis confirmed achieved power of 85.5%, exceeding the 80% adequacy threshold. Bootstrap 95% confidence intervals for the PLA+NK-75 median (6.0–9.0) showed no overlap with the control median CI (1.0–6.0), providing distribution-free confirmation of group separation. Model comparison using information criteria identified the Group × Time interaction model as optimal (AIC = 94.4, R² = 0.880), explaining 88% of variance — a substantial improvement over Group alone (R² = 0.323) or Time alone (R² = 0.454). PCA of the four Lane-Sandhu components revealed a dominant first principal component explaining 73.7% of variance, with all components loading positively (range: 0.352–0.556), representing an overall "healing factor." Internal consistency of the Lane-Sandhu scale was good (Cronbach's α = 0.866), supporting the reliability of the scoring instrument. Linear discriminant analysis achieved 73.3% classification accuracy in predicting group membership from component scores, with misclassifications occurring primarily between adjacent groups, consistent with the ordered treatment effect ( Figure 3 ). Table 1. Descriptive Statistics and Lane-Sandhu Component Scores by Group Parameter Control (n=11) PLA (n=4) PLA+NK-75 (n=15) p-valueᵃ Total Score 0.0144* Mean ± SD 4.00 ± 2.32 4.75 ± 3.59 7.20 ± 1.93 Median (IQR) 4.0 (4.00) 5.5 (4.25) 7.0 (3.00) Range 1–7 0–8 4–10 Bone Formation (0–4) 2.27 ± 1.01 2.25 ± 1.71 3.47 ± 0.64 0.0112* Proximal Union (0–2) 1.18 ± 0.87 1.25 ± 0.96 1.80 ± 0.41 0.1067 Distal Union (0–2) 0.55 ± 0.52 1.25 ± 0.96 1.47 ± 0.52 0.0047** Remodeling (0–2) 0.00 ± 0.00 0.00 ± 0.00 0.47 ± 0.74 0.0563 Score by Time Pointᵇ Week 4 1.0 0.0 5.2 Week 8 4.0 4.0 6.75 Week 12 5.0 7.0 9.0 Week 16 6.0 8.0 9.0 Week 20 7.0 NA 10.0 Healing Category, n (%) 0.0025 ᶜ No Healing / Poor (0–3) 4 (36.4%) 1 (25.0%) 1 (6.7%) Fair (4–5) 3 (27.3%) 1 (25.0%) 1 (6.7%) Good (6–7) 4 (36.4%) 1 (25.0%) 6 (40.0%) Excellent (8–10) 0 (0%) 1 (25.0%) 7 (46.7%) ᵃKruskal-Wallis test. ᵇMean total scores. ᶜFisher's Exact Test. *p < 0.05; **p < 0.01. NA = not available (no PLA observations at week 20). Table 2. Summary of Statistical Significance Across All Analytical Methods Analysis Method Test Statistic PLA+NK-75 vs Control PLA vs Control Overall p-value Primary Non-parametric Kruskal-Wallis χ² = 8.48 — — 0.0144* Dunn's Post-hoc (Bonferroni) Z = −2.870 0.0062** 0.7531 — Mann-Whitney U U = 28.0 0.0044** 0.5507 — Jonckheere-Terpstra JT = 207 — — 0.0021** Permutation Test (10,000 iter.) — — — 0.0080** Fisher's Exact (categories) — — — 0.0025** Sensitivity / Regression Models Linear Mixed Model t = 6.82 <0.001*** 0.273 — GEE (exchangeable) Wald χ² = 64.02 <0.001*** 0.005** — Ordinal Logistic Regression OR = 673.24 <0.001*** 0.304 — Poisson Regression RR = 1.91 <0.001*** 0.465 — Correlation Analyses Spearman ρ (Time vs Score) ρ = 0.705 — — <0.0001*** Linear Trend (ordered groups) r = 0.539 — — 0.0021** *p < 0.05; **p < 0.01; ***p < 0.001. GEE = Generalized Estimating Equations; RR = Rate Ratio; OR = Odds Ratio. Table 3. Effect Size Measures and Statistical Power Pairwise Effect Sizes PLA+NK-75 vs Control PLA+NK-75 vs PLA PLA vs Control Cliff's Delta 0.661 (Large) 0.433 (Medium) 0.227 (Small) Hedges' g 1.472 (Large) 1.011 (Large) 0.264 (Small) Glass's Δ 1.377 (Large) — 0.323 (Small) Probability of Superiority 83.0% — 61.4% Global Effect Sizes Value Interpretation Epsilon-squared (ε²) 0.292 Large Eta-squared (η²) 0.323 Large (32.3% variance explained) Cohen's f 0.643 Large Power and Model Fit Value Interpretation Achieved Power 85.5% Adequate (>80%) Best Model R² (Group × Time) 0.880 88% variance explained Cronbach's α (Lane-Sandhu) 0.866 Good internal consistency Bootstrap 95% CI: PLA+NK-75 median 6.0–9.0 Non-overlapping with Control CI Bootstrap 95% CI: Control median 1.0–6.0 — DISCUSSION The present study demonstrates that a PLA/NK-75 nanoclay biodegradable composite scaffold significantly enhances radiological bone healing in a rabbit critical-size ulnar defect model, with large effect sizes, accelerated milestone achievement, and exclusive remodeling activity. These findings contribute meaningfully to the growing body of evidence supporting nanoclay-augmented scaffolds for orthopaedic bone regeneration. The superior bone formation observed in the PLA+NK-75 group (mean bone formation score 3.47 ± 0.64 versus 2.27 ± 1.01 for controls) aligns with the findings of Lopresti et al. (2021), who demonstrated that incorporation of nanoclay into electrospun PLA nanofibers significantly enhanced pre-osteoblastic cell proliferation and improved surface wettability and mechanical properties compared to neat PLA scaffolds [15]. Our in vivo results thus provide biological confirmation of the in vitro advantages reported for PLA/nanoclay systems. Similarly, Sinha Ray (2012) reported in a comprehensive review that adding approximately 5 vol% clay to PLA improved storage modulus, tensile strength, and surface characteristics critical for tissue engineering, findings consistent with the 5 wt% nanoclay concentration employed in our composite [16]. The observation that remodeling activity was exclusively confined to the PLA+NK-75 group is particularly noteworthy. Neither the PLA-only nor the control group demonstrated any remodeling throughout the 20-week observation period, indicating that nanoclay augmentation was essential for healing to progress to its final maturation phase. This finding is consistent with the work of Ke et al. (2023), who showed that montmorillonite incorporation into keratin hydrogels dramatically enhanced osteogenic differentiation via the BMP-2/p-SMAD 1/5/8/RUNX2 signaling pathway and promoted superior bone regeneration in a rat cranial defect model [17]. The bioactive ions (silicon, magnesium, aluminium) released from montmorillonite-based nanoclays are known to stimulate osteoblastic activity and matrix mineralization, which may explain the advanced healing maturity observed exclusively in the nanoclay-containing group. Hu et al. (2020) provided important mechanistic insight by demonstrating that nanoclay's primary contribution to bone regeneration is through drug binding and sustained release of osteogenic growth factors rather than intrinsic osteoinductivity alone [18]. Although our scaffold was not loaded with exogenous growth factors, the sustained release of endogenous bioactive ions from the NK-75 montmorillonite may have functioned through an analogous mechanism of prolonged local bioactivity, supporting the progressive healing trajectory observed in the PLA+NK-75 group from week 4 through week 20 without plateau. The significant ordered trend (CONTROL < PLA < PLA+NK-75; Jonckheere-Terpstra p = 0.0021) validates the additive contribution of nanoclay to the polymer scaffold. This dose-response pattern is supported by Huang et al. (2019), who demonstrated that halloysite nanotube (a naturally occurring aluminosilicate nanoclay) incorporation into GelMA hydrogels significantly upregulated osteogenic gene expression and enhanced calvarial bone regeneration in rats compared to neat polymer scaffolds [19]. Li et al. (2024) further corroborated the osteogenic superiority of nanoclay-containing scaffolds, reporting that GelMA-nanoclay hydrogels enhanced osteogenesis, angiogenesis, and immunomodulation in bone defects [20]. Our use of the Lane-Sandhu scoring system is consistent with the methodology employed by Basaran et al. (2022), who used a modified Lane-Sandhu system to evaluate bone healing in a rabbit ulnar segmental defect model [21]. However, whereas their biocomposite scaffold without mesenchymal stem cells achieved satisfactory regeneration by 6 weeks, our PLA+NK-75 composite achieved good healing (score ≥6) as early as week 4, suggesting that nanoclay-enhanced scaffolds may accelerate early-phase healing even without exogenous cellular supplementation. The 83% probability of superiority of PLA+NK-75 over controls and the 88% variance explained by the Group × Time interaction model underscore both the magnitude and reliability of the treatment effect. Salehi et al. (2024) similarly reported that PLA-based nanocomposite scaffolds incorporating bredigite nanoparticles achieved near-complete calvarial bone recovery in rats within 8 weeks [22], supporting the principle that bioactive nanofiller incorporation into PLA matrices substantially amplifies regenerative capacity. Furthermore, Zhang et al. (2021) demonstrated that even 1% w/v nanosilicate incorporation into silk fibroin hydrogels was sufficient to enhance dual-lineage differentiation and promote osteochondral regeneration in rabbit defects [23], suggesting that nanoclays exert potent biological effects at relatively low concentrations. Several limitations of this study warrant consideration. The unequal group sizes, particularly the small PLA-only group (n = 4), limit the statistical power for PLA versus control comparisons, although the primary PLA+NK-75 versus control comparison achieved adequate power (85.5%). The study relied exclusively on radiological assessment using the Lane-Sandhu system without quantitative histomorphometry or micro-CT analysis. The rabbit ulnar model, while well-established, does not replicate the biomechanical loading environment of human long bones. Future studies should incorporate histomorphometric quantification, micro-CT volumetric analysis, and biomechanical testing, along with dose-optimization of nanoclay concentration and evaluation in larger animal models to further validate these findings prior to clinical translation. CONCLUSION The PLA/NK-75 biodegradable nanoclay composite scaffold demonstrated statistically significant and clinically meaningful superiority over both PLA alone and untreated empty defect controls in promoting radiological bone healing in a rabbit critical-size ulnar defect model. The composite achieved the highest Lane-Sandhu scores at every time point, with large effect sizes, strong probability of superiority over controls, and markedly accelerated healing milestones. Remodeling, the final phase of bone regeneration, was exclusively observed in the PLA+NK-75 group, indicating the composite supports healing to a degree of maturity unattainable by PLA alone or natural repair. A significant ordered trend validated the hypothesis that nanoclay augmentation provides additive benefit to the biodegradable polymer scaffold. These results were robust across multiple independent statistical methods, strengthening causal inference. The findings support PLA/NK-75 nanoclay composite as a promising, cost-effective scaffold material for orthopaedic bone regeneration that warrants further preclinical investigation with histomorphometric, micro-CT, and biomechanical endpoints, dose-optimization studies, and eventual translation toward larger animal models. Abbreviations AIC Akaike information criterion ARRIVE Animal Research:Reporting of In Vivo Experiments BIC Bayesian information criterion BMP-2 Bone morphogenetic protein 2 CI Confidence interval CPCSEA Committee for the Purpose of Control and Supervision of Experiments on Animals EDAX Energy-dispersive X-ray analysis FDA Food and Drug Administration GEE Generalised estimating equations GelMA Gelatin methacryloyl H&E Haematoxylin and eosin IAEC Institutional Animal Ethics Committee IQR Interquartile range NK-75 Nanoclay (organically modified montmorillonite) OR Odds ratio PCA Principal component analysis PLA Polylactic acid RR Rate ratio RUNX2 Runt-related transcription factor 2 SD Standard deviation Declarations Ethics approval and consent to participate This study was approved by the Institutional Animal Ethics Committee (IAEC), Institute of Medical Sciences, Banaras Hindu University, Varanasi, India (Approval No. Dean/2023/IAEC/6193, dated 10.08.2023; CPCSEA Registration No. 542/GO/ReBi//S/02/CCSEA, dated 17.07.2023). All experimental procedures were conducted in accordance with the guidelines of the Committee for the Purpose of Control and Supervision of Experiments on Animals (CPCSEA), Government of India. Consent for publication: Not applicable. Clinical trial registration: Not applicable. Availability of data and materials The datasets generated and/or analysed during the current study are available from the corresponding author on reasonable request. Competing interests The authors declare that they have no competing interests. Funding This research did not receive any specific grant from funding agencies in the public, commercial, or not-for-profit sectors. Authors' contributions AR: Conceptualisation, Methodology, Investigation, Formal analysis, Data curation, Visualisation, Writing — original draft. AS: Conceptualisation, Methodology, Supervision, Project administration, Resources, Validation, Writing — review and editing. PM: Methodology, Resources, Supervision, Writing — review and editing. SY: Investigation, Validation, Writing — review and editing. AG: Investigation (histological evaluation and interpretation of H&E, Toluidine Blue O, and Von Kossa stained section, Validation, Writing — review and editing. SM: Methodology, Investigation, Resources. AnR: Investigation, Data curation, Writing — review and editing. SRN: Investigation, Data curation. SJ: Investigation, Data curation. All authors read and approved the final manuscript. Acknowledgements: Not applicable. References Rodham PL, Giannoudis VP, Kanakaris NK, Giannoudis PV. Biological aspects to enhance fracture healing. EFORT Open Rev. 2023;8(5):264–282. doi:10.1530/EOR-23-0047 Pereira HF, Cengiz IF, Silva FS, Reis RL, Oliveira JM. Scaffolds and coatings for bone regeneration. J Mater Sci Mater Med. 2020;31(3):27. doi:10.1007/s10856-020-06364-y Busch A, Jäger M. Synthetic bone replacement substances. Orthopädie (Heidelb). 2022;51(12):1023–1032. doi:10.1007/s00132-022-04319-5 Wang Y, Wang J, Gao R, et al. Biomimetic glycopeptide hydrogel coated PCL/nHA scaffold for enhanced cranial bone regeneration via macrophage M2 polarization-induced osteo-immunomodulation. Biomaterials. 2022;285:121538. doi:10.1016/j.biomaterials.2022.121538 Arif U, Haider S, Haider A, et al. Biocompatible polymers and their potential biomedical applications: a review. Curr Pharm Des. 2019;25(34):3608–3619. doi:10.2174/1381612825999191011105148 Alavi MS, Memarpour S, Pazhohan-Nezhad H, Salimi Asl A, Moghbeli M, Shadmanfar S, Saburi E. Applications of poly(lactic acid) in bone tissue engineering: a review article. Artif Organs. 2023;47(9):1423–1430. doi:10.1111/aor.14612 Tyler B, Gullotti D, Mangraviti A, Utsuki T, Brem H. Polylactic acid (PLA) controlled delivery carriers for biomedical applications. Adv Drug Deliv Rev. 2016;107:163–175. doi:10.1016/j.addr.2016.06.018 Mohan A, Girdhar M, Kumar R, et al. Polyhydroxybutyrate-based nanocomposites for bone tissue engineering. Pharmaceuticals (Basel). 2021;14(11):1163. doi:10.3390/ph14111163 Katti KS, Jasuja H, Jaswandkar SV, Mohanty S, Katti DR. Nanoclays in medicine: a new frontier of an ancient medical practice. Mater Adv. 2022;3(20):7484–7500. doi:10.1039/d2ma00528j Yao Q, Fuglsby KE, Zheng X, Sun H. Nanoclay-functionalized 3D nanofibrous scaffolds promote bone regeneration. J Mater Chem B. 2020;8(17):3842–3851. doi:10.1039/c9tb02814e Zheng X, Zhang X, Wang Y, et al. Hypoxia-mimicking 3D bioglass-nanoclay scaffolds promote endogenous bone regeneration. Bioact Mater. 2021;6(10):3485–3495. doi:10.1016/j.bioactmat.2021.03.011 Erezuma I, Lukin I, Pimenta-Lopes C, et al. Nanoclay-reinforced HA/alginate scaffolds as cell carriers and SDF-1 delivery-platforms for bone tissue engineering. Int J Pharm. 2022;623:121895. doi:10.1016/j.ijpharm.2022.121895 Kapusetti G, Misra N, Singh V, et al. Bone cement based nanohybrid as a super biomaterial for bone healing. J Mater Chem B. 2014;2(25):3984–3997. doi:10.1039/C4TB00501E Senapati S, Mahanta AK, Kumar S, Maiti P. Controlled drug delivery vehicles for cancer treatment and their performance. Signal Transduct Target Ther. 2018;3:7. doi:10.1038/s41392-017-0004-3 Lopresti F, Carfì Pavia F, Ceraulo M, Capuana E, Brucato V, Ghersi G, et al. Physical and biological properties of electrospun poly(d,l-lactide)/nanoclay and poly(d,l-lactide)/nanosilica nanofibrous scaffold for bone tissue engineering. J Biomed Mater Res A. 2021;109(11):2120–2136. doi:10.1002/jbm.a.37199 Sinha Ray S. Polylactide-based bionanocomposites: a promising class of hybrid materials. Acc Chem Res. 2012;45(10):1710–1720. doi:10.1021/ar3000376 Ke Y, Wu J, Ye Y, Zhang X, Gu T, Wang Y, et al. Feather keratin-montmorillonite nanocomposite hydrogel promotes bone regeneration by stimulating the osteogenic differentiation of endogenous stem cells. Int J Biol Macromol. 2023;243:125330. doi:10.1016/j.ijbiomac.2023.125330 Hu J, Miszuk JM, Stein KM, Sun H. Nanoclay promotes mouse cranial bone regeneration mainly through modulating drug binding and sustained release. Appl Mater Today. 2020;21:100860. doi:10.1016/j.apmt.2020.100860 Huang K, Ou Q, Xie Y, Chen X, Fang Y, Huang C, et al. Halloysite nanotube based scaffold for enhanced bone regeneration. ACS Biomater Sci Eng. 2019;5(8):4037–4047. doi:10.1021/acsbiomaterials.9b00277 Li H, Mao B, Zhong J, Li X, Sang H. Localized delivery of metformin via 3D printed GelMA-Nanoclay hydrogel scaffold for enhanced treatment of diabetic bone defects. J Orthop Translat. 2024;47:249–260. doi:10.1016/j.jot.2024.06.013 Basaran SH, Bayrak A, Tanrıverdi G, Tanriverdi B, Avkan MC. Partial load-bearing rabbit ulnar segmental defects are regenerated with biocompatible grafts with or without bone marrow-derived mesenchymal stem cells. Ulus Travma Acil Cerrahi Derg. 2022;28(8):1066–1072. doi:10.14744/tjtes.2021.64569 Salehi S, Ghomi H, Hassanzadeh-Tabrizi SA, Koupaei N, Khodaei M. 3D printed polylactic acid/polyethylene glycol/bredigite nanocomposite scaffold enhances bone tissue regeneration via promoting osteogenesis and angiogenesis. Int J Biol Macromol. 2024;281(Pt 1):136160. doi:10.1016/j.ijbiomac.2024.136160 Zhang W, Zhang Y, Zhang A, Ling C, Sheng R, Li X, et al. Enzymatically crosslinked silk-nanosilicate reinforced hydrogel with dual-lineage bioactivity for osteochondral tissue engineering. Mater Sci Eng C Mater Biol Appl. 2021;127:112215. doi:10.1016/j.msec.2021.112215 Additional Declarations No competing interests reported. Cite Share Download PDF Status: Under Review Version 1 posted Reviews received at journal 15 May, 2026 Reviewers agreed at journal 14 May, 2026 Reviewers agreed at journal 12 May, 2026 Reviewers invited by journal 25 Feb, 2026 Editor assigned by journal 25 Feb, 2026 Editor invited by journal 23 Feb, 2026 Submission checks completed at journal 21 Feb, 2026 First submitted to journal 21 Feb, 2026 You are reading this latest preprint version Research Square lets you share your work early, gain feedback from the community, and start making changes to your manuscript prior to peer review in a journal. As a division of Research Square Company, we’re committed to making research communication faster, fairer, and more useful. We do this by developing innovative software and high quality services for the global research community. Our growing team is made up of researchers and industry professionals working together to solve the most critical problems facing scientific publishing. Also discoverable on Platform About Our Team In Review Editorial Policies Advisory Board Help Center Resources Author Services Accessibility API Access RSS feed Manage Cookie Preferences © Research Square 2026 | ISSN 2693-5015 (online) Privacy Policy Terms of Service Do Not Sell My Personal Information {"props":{"pageProps":{"initialData":{"identity":"rs-8882301","acceptedTermsAndConditions":true,"allowDirectSubmit":false,"archivedVersions":[],"articleType":"Research Article","associatedPublications":[],"authors":[{"id":596823573,"identity":"93a5ee62-3049-4a62-ab4f-cbc0d5a9b458","order_by":0,"name":"Ashish Ranjan","email":"","orcid":"","institution":"Institute of Medical Sciences, Banaras Hindu University","correspondingAuthor":false,"prefix":"","firstName":"Ashish","middleName":"","lastName":"Ranjan","suffix":""},{"id":596823576,"identity":"98e15b29-5fa1-483b-bbff-cb8f9aee40fe","order_by":1,"name":"Ajit Singh","email":"data:image/png;base64,iVBORw0KGgoAAAANSUhEUgAAAZAAAAAyAQMAAABI0h/eAAAABlBMVEX///8AAABVwtN+AAAACXBIWXMAAA7EAAAOxAGVKw4bAAABD0lEQVRIiWNgGAWjYFCCgw0MDAZAxN6QcOBDBVCAmbmBkJbGBrAWngMPH844A9LCSEgLA0SFgUTiY2PeNoQATsDfeLj94Y+CO3bbGZLTpHnn1UbztwO1/KjYhlOLxIGDjc08Bs+SdzYcS5Ocu+147ozDjA2MPWdu47YGpIXB4HCywcGeNIm3247lNgC1MDO24dYiD9TS+AOk5TD/NwneOcdy5xPSYgDU0sBjcNjO4BhDsiFvQ03uBkJaDIFaZgO1JBicYUh8OOPYgdyNQC0H8flF7sbxBx9//Dlsb3D/ATAqa+py550/fPDBjwo83pc4AKYSGyDcw5Awwa0eCPghau2h3Dq8ikfBKBgFo2BkAgD9Xm6gmbsHAwAAAABJRU5ErkJggg==","orcid":"","institution":"Institute of Medical Sciences, Banaras Hindu University","correspondingAuthor":true,"prefix":"","firstName":"Ajit","middleName":"","lastName":"Singh","suffix":""},{"id":596823578,"identity":"91983889-9689-43b1-bbd9-4db5bf1595fb","order_by":2,"name":"Pralay Maiti","email":"","orcid":"","institution":"Indian Institute of Technology (Banaras Hindu University)","correspondingAuthor":false,"prefix":"","firstName":"Pralay","middleName":"","lastName":"Maiti","suffix":""},{"id":596823580,"identity":"10cf01a9-1a13-4d12-95e4-cad253e9a41d","order_by":3,"name":"Sanjay Yadav","email":"","orcid":"","institution":"Institute of Medical Sciences, Banaras Hindu University","correspondingAuthor":false,"prefix":"","firstName":"Sanjay","middleName":"","lastName":"Yadav","suffix":""},{"id":596823582,"identity":"6989cb7e-e97f-4bf2-819a-05fb84e4a1db","order_by":4,"name":"Amrita Ghosh","email":"","orcid":"","institution":"Institute of Medical Sciences, Banaras Hindu University","correspondingAuthor":false,"prefix":"","firstName":"Amrita","middleName":"","lastName":"Ghosh","suffix":""},{"id":596823583,"identity":"46a54ca6-9b6b-4437-bb5c-32b557152fc7","order_by":5,"name":"Swapan Maity","email":"","orcid":"","institution":"Indian Institute of Technology (Banaras Hindu University)","correspondingAuthor":false,"prefix":"","firstName":"Swapan","middleName":"","lastName":"Maity","suffix":""},{"id":596823584,"identity":"31589a94-1c94-499c-be8b-7ea999a31352","order_by":6,"name":"Anshul Raj","email":"","orcid":"","institution":"Institute of Medical Sciences, Banaras Hindu University","correspondingAuthor":false,"prefix":"","firstName":"Anshul","middleName":"","lastName":"Raj","suffix":""},{"id":596823585,"identity":"93a425a3-1dd5-48bf-aa44-b63c0a81e302","order_by":7,"name":"Soumya Ranjan Nayak","email":"","orcid":"","institution":"Institute of Medical Sciences, Banaras Hindu University","correspondingAuthor":false,"prefix":"","firstName":"Soumya","middleName":"Ranjan","lastName":"Nayak","suffix":""},{"id":596823587,"identity":"5cd3b9db-0cad-41ed-92b4-5c7faab8c298","order_by":8,"name":"Samay Jaiswal","email":"","orcid":"","institution":"Institute of Medical Sciences, Banaras Hindu University","correspondingAuthor":false,"prefix":"","firstName":"Samay","middleName":"","lastName":"Jaiswal","suffix":""}],"badges":[],"createdAt":"2026-02-14 19:53:15","currentVersionCode":1,"declarations":"","doi":"10.21203/rs.3.rs-8882301/v1","doiUrl":"https://doi.org/10.21203/rs.3.rs-8882301/v1","draftVersion":[],"editorialEvents":[],"editorialNote":"","failedWorkflow":false,"files":[{"id":104007657,"identity":"12c7643f-2b04-4a9a-95e8-dd6f6ec9965c","added_by":"auto","created_at":"2026-03-05 15:20:08","extension":"png","order_by":1,"title":"Figure 1","display":"","copyAsset":false,"role":"figure","size":1551872,"visible":true,"origin":"","legend":"\u003cp\u003eSerial radiological and histological evaluation of the control (untreated empty defect) group. \u003cstrong\u003e(A)\u003c/strong\u003e Anteroposterior radiographs of the right ulna at 0, 4, 8, 12, and 16 weeks post-surgery showing the progression of bone healing at the mid-diaphyseal osteotomy site. \u003cstrong\u003e(B)\u003c/strong\u003e Photomicrograph of the defect site (hematoxylin and eosin staining) showing woven bone with osteocytes (arrows). Scale bar shown. Scale bar = 40 μm.\u003c/p\u003e","description":"","filename":"image1.png","url":"https://assets-eu.researchsquare.com/files/rs-8882301/v1/50f4b3920c557807a587cbf6.png"},{"id":104007662,"identity":"fed55784-6b1a-4a1d-930d-bd83383d4106","added_by":"auto","created_at":"2026-03-05 15:20:10","extension":"png","order_by":2,"title":"Figure 2","display":"","copyAsset":false,"role":"figure","size":1986515,"visible":true,"origin":"","legend":"\u003cp\u003eSerial radiological and histological evaluation of the PLA-only scaffold group. \u003cstrong\u003e(A)\u003c/strong\u003eAnteroposterior radiographs of the ulna at 0, 4, 8, 12, 16, and 20 weeks post-surgery showing the progression of bone healing at the mid-diaphyseal osteotomy site. \u003cstrong\u003e(B)\u003c/strong\u003e Photomicrograph of the defect site (hematoxylin and eosin staining) showing cortical bone and periosteum. Scale bar shown. Scale bar = 40 μm.\u003c/p\u003e","description":"","filename":"image2.png","url":"https://assets-eu.researchsquare.com/files/rs-8882301/v1/e3c80fdde81fdf40d656d18e.png"},{"id":104007658,"identity":"79f69ccb-7e1b-44b1-b56a-7e6885d143a8","added_by":"auto","created_at":"2026-03-05 15:20:08","extension":"png","order_by":3,"title":"Figure 3","display":"","copyAsset":false,"role":"figure","size":1534207,"visible":true,"origin":"","legend":"\u003cp\u003eSerial radiological and histological evaluation of the PLA+NK-75 composite scaffold group. \u003cstrong\u003e(A)\u003c/strong\u003e Anteroposterior radiographs of the ulna at 0, 4, 8, 12, and 16 weeks post-surgery showing the progression of bone healing at the mid-diaphyseal osteotomy site. \u003cstrong\u003e(B)\u003c/strong\u003e Photomicrograph of the defect site (hematoxylin and eosin staining) showing osteocytes and marrow elements. Scale bar shown. Scale bar = 40 μm.\u003c/p\u003e","description":"","filename":"image3.png","url":"https://assets-eu.researchsquare.com/files/rs-8882301/v1/3226946c2cc7f426d4817cb1.png"},{"id":104402291,"identity":"eea9b9e5-0949-489e-8b07-49ee19d9e88c","added_by":"auto","created_at":"2026-03-11 12:14:59","extension":"png","order_by":4,"title":"Figure 4","display":"","copyAsset":false,"role":"figure","size":125750,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eTemporal healing trajectories by group.\u003c/strong\u003e Line graph depicting mean Lane-Sandhu total scores at each time point (weeks 4, 8, 12, 16, and 20) for PLA+NK-75 (solid line, circles), PLA (dashed line, triangles), and Control (dotted line, squares) groups. Error bars represent standard deviation.\u003c/p\u003e","description":"","filename":"image4.png","url":"https://assets-eu.researchsquare.com/files/rs-8882301/v1/1e07b6662c582a7008e67a27.png"},{"id":104007660,"identity":"c7de890d-78b3-4ada-9e6d-7b4a774fd40d","added_by":"auto","created_at":"2026-03-05 15:20:09","extension":"png","order_by":5,"title":"Figure 5","display":"","copyAsset":false,"role":"figure","size":1910980,"visible":true,"origin":"","legend":"\u003cp\u003eComparative serial radiographs across treatment groups. Anteroposterior radiographs of the rabbit ulna at 0 days, 8 weeks, and 16 weeks post-surgery for Control \u003cstrong\u003e(A–C)\u003c/strong\u003e, PLA \u003cstrong\u003e(D–F)\u003c/strong\u003e, and PLA+NK-75 \u003cstrong\u003e(G–I)\u003c/strong\u003e groups. Rows represent treatment groups; columns represent time points.\u003c/p\u003e","description":"","filename":"image5.png","url":"https://assets-eu.researchsquare.com/files/rs-8882301/v1/7cccc1d1f6bef4818b517f13.png"},{"id":104007659,"identity":"678078a1-595c-40c8-a7c0-98188ad6d5e9","added_by":"auto","created_at":"2026-03-05 15:20:09","extension":"png","order_by":6,"title":"Figure 6","display":"","copyAsset":false,"role":"figure","size":2966419,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eComparative histological evaluation across treatment groups and time points. Photomicrographs of the osteotomy site (hematoxylin and eosin staining). Top row: Sections at a single time point from Control (A), PLA (B), and PLA+NK-75 (C) groups. Bottom row: Sections from the PLA+NK-75 group at 4 weeks (D), 12 weeks (E), and 16 weeks (F) post-surgery. Scale bar = 40 μm.\u003c/strong\u003e\u003c/p\u003e","description":"","filename":"image6.png","url":"https://assets-eu.researchsquare.com/files/rs-8882301/v1/37efb112d80d058bd295fd2f.png"},{"id":104408355,"identity":"6b6dea57-171d-4337-bb7c-f1b11e0b5ef8","added_by":"auto","created_at":"2026-03-11 12:42:17","extension":"pdf","order_by":0,"title":"","display":"","copyAsset":false,"role":"manuscript-pdf","size":16095558,"visible":true,"origin":"","legend":"","description":"","filename":"manuscript.pdf","url":"https://assets-eu.researchsquare.com/files/rs-8882301/v1/e7dda830-66fc-4cb6-bca1-c127c3b8f33b.pdf"}],"financialInterests":"No competing interests reported.","formattedTitle":"Efficacy of a novel polylactic acid/nanoclay (NK-75) biodegradable composite scaffold in healing critical-size bone defects: an experimental study in a rabbit ulnar model","fulltext":[{"header":"INTRODUCTION","content":"\u003cp\u003eCritical-size bone defects arising from trauma, tumour resection, infection debridement, and revision surgery remain among the most challenging problems in contemporary orthopaedic practice [1,2]. Fracture nonunion complicates an estimated 5\u0026ndash;10% of all fractures and imposes a substantial clinical and socioeconomic burden through prolonged disability, repeated surgical interventions, and diminished quality of life [1]. Autologous bone grafting continues to be regarded as the gold standard owing to its combined osteogenic, osteoinductive, and osteoconductive properties; however, its utility is constrained by limited graft volume, donor-site morbidity, and the requirement for a second operative site [2,3]. These limitations have stimulated considerable research into synthetic scaffold-based alternatives that can be manufactured reproducibly and made available off the shelf [3].\u003c/p\u003e \u003cp\u003eBiodegradable polymer scaffolds have emerged as a promising bone tissue engineering strategy, providing temporary mechanical support while gradually resorbing and being replaced by regenerating bone [4,5]. Among the biodegradable polymers, polylactic acid (PLA) has attracted particular attention because of its established biocompatibility, FDA approval for clinical use, tunable degradation kinetics, and favourable mechanical properties [6,7]. Nevertheless, PLA exhibits several recognised shortcomings, including limited osteoconductivity, a relatively hydrophobic surface that impedes cellular adhesion, and the generation of acidic degradation products that can provoke local inflammation and impair osteoblast function [6].\u003c/p\u003e \u003cp\u003eTo address these limitations, the incorporation of nanoscale inorganic fillers into polymer matrices has been explored extensively [8]. Nanoclays, particularly montmorillonite-based layered aluminosilicates, represent an attractive class of bioactive nanofillers owing to their high surface area, cation exchange capacity, and established safety in biomedical applications [9]. Recent investigations have demonstrated that nanoclay-functionalised scaffolds significantly enhance osteogenic differentiation of mesenchymal stem cells in vitro and promote bone regeneration in vivo through multiple mechanisms, including mechanical reinforcement, sustained release of bioactive ions (silicon, magnesium), buffering of acidic degradation byproducts, and improved surface hydrophilicity for cell attachment [10\u0026ndash;12]. Furthermore, polymer-nanoclay nanocomposites have shown promise in bone cement and drug delivery systems [13,14].\u003c/p\u003e \u003cp\u003eDespite these encouraging findings, in vivo evaluation of PLA/nanoclay composites specifically employing organically modified montmorillonite (NK-75) in an orthopaedic bone defect model has not been reported. The present study aimed to evaluate the efficacy of a novel PLA/NK-75 biodegradable composite scaffold in healing critical-size bone defects compared to PLA alone and untreated empty defect controls in a rabbit ulnar model, with the hypothesis that nanoclay augmentation would produce superior radiological healing in an ordered dose-response pattern (CONTROL\u0026thinsp;\u0026lt;\u0026thinsp;PLA\u0026thinsp;\u0026lt;\u0026thinsp;PLA\u0026thinsp;+\u0026thinsp;NK-75).\u003c/p\u003e"},{"header":"MATERIALS AND METHODS","content":"\u003cp\u003e\u003cstrong\u003eEthical Approval\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThis study was approved by the Institutional Animal Ethics Committee (IAEC), Institute of Medical Sciences, Banaras Hindu University, Varanasi (Registration No. 542/GO/ReBi/S/02/CPCSEA, dated 26.05.2017), and conducted in accordance with the guidelines of the Committee for the Purpose of Control and Supervision of Experiments on Animals (CPCSEA), Government of India. The study is reported following the ARRIVE 2.0 guidelines for animal research.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eScaffold Preparation\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eMaterials\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003ePolylactic acid (PLA) served as the biodegradable polymer matrix. NK-75 nanoclay, an organically modified montmorillonite (ion-exchanged with dimethyl dihydrogenated tallow ammonium; cation exchange capacity 70 mequiv. per 100 g), was selected as the bioactive nanofiller. Elemental composition of NK-75 was confirmed using energy-dispersive X-ray analysis (EDAX). Scaffold preparation was performed in collaboration with the School of Material Sciences and Technology, IIT-BHU.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eComposite Fabrication\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003ePLA+NK-75 nanohybrid composites were prepared by solution-mixing PLA with 5 wt% NK-75 nanoclay using a handheld mixer for 10 minutes to achieve uniform nanoclay dispersion. The composite was molded into cylindrical scaffolds of 3 mm length matching the planned defect size. PLA-only scaffolds were prepared using an identical protocol without nanoclay addition. All scaffolds were sterilized prior to surgical implantation.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAnimal Model and Experimental Design\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAnimals\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eSkeletally mature New Zealand White rabbits (\u003cem\u003eOryctolagus cuniculus\u003c/em\u003e), aged 6 months and weighing up to 3 kg, of both sexes, were obtained from the institutional animal facility, IMS-BHU. Animals were housed under standard conditions with ad libitum access to food and water, 12-hour light-dark cycles, and were acclimatized for one week prior to surgery.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eStudy Groups and Sample Size\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe study comprised 30 experimental observations distributed across three groups. The PLA+NK-75 group (n=15 observations) served as the primary experimental arm. The PLA-only group (n=4 observations) served as the polymer control to isolate the contribution of nanoclay. The untreated empty defect group (n=11 observations) served as the negative control. Animals underwent bilateral ulnar osteotomy, with each limb serving as an independent observation. Animals were sequentially sacrificed at five time points (weeks 4, 8, 12, 16, and 20), with the distribution reflecting the primary focus on early-to-mid healing phases: week 4 (n=9), week 8 (n=8), week 12 (n=6), week 16 (n=5), and week 20 (n=2). Unequal group sizes were accounted for using non-parametric statistical methods and confirmed with post-hoc power analysis.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eSurgical Procedure\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eRabbits were anesthetized with an intramuscular gluteal injection of ketamine (30 mg/kg) and midazolam (1 mg/kg). Bilateral forearms were shaved and disinfected with povidone-iodine. Through a direct approach to the mid-diaphysis of the ulna, a 3 mm segmental osteotomy was performed extraperiosteally, creating a critical-size defect. In treatment groups, a 3 mm scaffold (PLA+NK-75 or PLA-only) was press-fit into the defect gap. Control limbs received no scaffold, leaving the defect empty. Wound closure was performed in layers using absorbable (Vicryl) and non-absorbable (Nylon/Prolene) sutures. Three postoperative doses of gentamicin (10 mg) were administered for infection prophylaxis. Animals were allowed unrestricted cage activity postoperatively.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eSacrifice and Specimen Harvest\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eAt each designated time point, rabbits were humanely euthanized by intravenous injection of sodium pentobarbital (\u0026gt;100 mg/kg). Bilateral ulnar specimens were harvested en bloc with surrounding soft tissue for radiological assessment.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eOutcome Assessment\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eRadiological Scoring — Lane-Sandhu System\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eAnteroposterior radiographs of harvested specimens were obtained at each time point . Radiological healing was assessed using the Lane-Sandhu scoring system, a validated semi-quantitative grading scale comprising four components: bone formation (0–4 points: 0=no new bone, 1=25% of defect filled, 2=50%, 3=75%, 4=100%), proximal union (0–2 points: 0=no union, 1=partial, 2=complete), distal union (0–2 points: same criteria), and remodeling (0–2 points: 0=none, 1=recanalization of medullary canal, 2=cortical remodeling). The total score ranged from 0 (no healing) to 10 (complete healing with remodeling). Total scores were further categorized as: No Healing (0), Poor (1–3), Fair (4–5), Good (6–7), and Excellent (8–10).\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eHistological Assessment\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eFollowing radiological assessment, specimens were processed for histological evaluation. Sections from the implant site and adjacent tissues were obtained for gross examination (bone status, soft tissue status, wound integrity) and microscopic evaluation using hematoxylin and eosin (H\u0026amp;E) for general cellular architecture, Toluidine Blue O for demonstration of mineralized bone, osteoid seams, and osteoblast/osteoclast identification, and Von Kossa staining for mineralization assessment. Osteoblastic activity and periscaffold tissue response were evaluated qualitatively.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eStatistical Analysis\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eAll analyses were performed using IBM SPSS Statistics version 29.0 (IBM Corp., Armonk, NY, USA). Descriptive statistics are reported as mean ± standard deviation (SD), median, interquartile range (IQR), and range. The primary analysis employed the Kruskal-Wallis test for overall group comparison, followed by Dunn's post-hoc test with Bonferroni correction and Mann-Whitney U tests for pairwise comparisons. Fisher's Exact Test assessed the association between treatment group and categorical healing outcomes. The Jonckheere-Terpstra test evaluated the a priori ordered alternative hypothesis (CONTROL \u0026lt; PLA \u0026lt; PLA+NK-75). Spearman's rank correlation assessed the relationship between time and healing scores.\u003c/p\u003e\n\u003cp\u003eSensitivity analyses included a linear mixed model with animal ID as a random effect, generalized estimating equations (GEE) with exchangeable correlation structure, ordinal logistic regression (cumulative link model) for ordered healing categories, and Poisson regression for count-modeled total scores. Negative binomial regression was performed to assess overdispersion. Robust regression using M-estimation was performed to evaluate outlier influence.\u003c/p\u003e\n\u003cp\u003eEffect sizes were calculated using Cliff's delta (non-parametric), Hedges' g and Glass's Δ (standardized mean differences), epsilon-squared and eta-squared (variance explained), and Cohen's f. The common language effect size (probability of superiority) was computed for each pairwise comparison. Bootstrap confidence intervals for group medians were generated using 10,000 resampling iterations. Permutation testing (10,000 iterations) provided exact p-values free from distributional assumptions.\u003c/p\u003e\n\u003cp\u003eInternal consistency of the Lane-Sandhu scale was evaluated using Cronbach's alpha. Principal component analysis (PCA) assessed the dimensional structure of the four scoring components. Post-hoc power analysis determined achieved statistical power. Model comparison using Akaike Information Criterion (AIC) and Bayesian Information Criterion (BIC) identified the optimal predictor combination. A two-sided p-value \u0026lt; 0.05 was considered statistically significant.\u003c/p\u003e"},{"header":"RESULTS","content":"\u003cp\u003e\u003cstrong\u003eSample Characteristics and Overall Healing Scores\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThirty observations across three groups and five time points were analyzed (Figure 1,2 and 3). No perioperative mortality, wound infection, or scaffold-related adverse events were recorded. The descriptive statistics and component-level Lane-Sandhu scores are presented in \u003cstrong\u003eTable 1\u003c/strong\u003e.\u003c/p\u003e\n\u003cp\u003eThe PLA+NK-75 group demonstrated the highest mean total score of 7.20 \u0026plusmn; 1.93, exceeding both PLA (4.75 \u0026plusmn; 3.59) and control (4.00 \u0026plusmn; 2.32). The PLA+NK-75 group was the only group to achieve the maximum possible score of 10, while the control group\u0026apos;s maximum was limited to 7. Across all four Lane-Sandhu components, PLA+NK-75 showed consistent superiority. Bone formation was highest in PLA+NK-75 (3.47 \u0026plusmn; 0.64) compared to control (2.27 \u0026plusmn; 1.01) and PLA (2.25 \u0026plusmn; 1.71). Distal union showed the most pronounced intergroup differences (PLA+NK-75: 1.47 \u0026plusmn; 0.52 vs. control: 0.55 \u0026plusmn; 0.52). Notably, remodeling activity was exclusively observed in the PLA+NK-75 group (0.47 \u0026plusmn; 0.74), with no remodeling in either control or PLA groups throughout the entire observation period, indicating that only the nanoclay-enhanced composite achieved sufficient healing maturity to initiate the final phase of bone regeneration.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eTemporal Healing Progression and Categorical Outcomes\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eA clear temporal progression was observed, with overall mean scores increasing from 3.22 \u0026plusmn; 2.44 at week 4 to 8.50 \u0026plusmn; 2.12 at week 20 (Spearman \u0026rho; = 0.705, p \u0026lt; 0.0001). Group-wise temporal trajectories, illustrated in \u003cstrong\u003eFigure 4\u003c/strong\u003e, revealed striking differences. PLA+NK-75 demonstrated superior scores at every time point \u0026mdash; 5.2 at week 4 rising to 10.0 by week 20 \u0026mdash; whereas controls progressed from 1.0 to only 7.0 over the same period. PLA+NK-75 achieved a score of 9.0 by week 12, a level the control group never attained even by week 20. Comparative serial radiographs across all three groups at 0 days, 8 weeks, and 16 weeks further illustrated the superior healing trajectory in PLA+NK-75 relative to PLA and control groups (Figure 5).\u003c/p\u003e\n\u003cp\u003eTime-to-milestone analysis further highlighted the accelerated healing in PLA+NK-75. Good healing (score \u0026ge;6) was first achieved at week 4 in PLA+NK-75, compared to week 8 in controls and week 12 in PLA. Excellent healing (score \u0026ge;8) was attained by 7 PLA+NK-75 observations beginning at week 8, while controls never reached this threshold. The Friedman test confirmed significant within-group time progression only in PLA+NK-75 (\u0026chi;\u0026sup2; = 11.92, p = 0.018), indicating sustained progressive healing without plateau \u0026mdash; a clinically desirable property for bone regeneration scaffolds.\u003c/p\u003e\n\u003cp\u003eCategorical healing outcomes demonstrated marked intergroup differences. Excellent healing (score 8\u0026ndash;10) was achieved in 46.7% (7/15) of PLA+NK-75 observations, compared to 25.0% (1/4) in PLA and 0% (0/11) in controls. Conversely, poor healing (score 1\u0026ndash;3) was predominantly observed in controls (36.4%, 4/11), with only a single instance in PLA+NK-75 (6.7%). Fisher\u0026apos;s Exact Test confirmed a significant association between treatment group and healing category (p = 0.0025).\u003c/p\u003e\n\u003cp\u003eHistological evaluation corroborated the radiological findings. Comparative histological sections across groups and time points further demonstrated the superior tissue quality and progressive maturation in the PLA+NK-75 group relative to PLA and control groups (Figure 6).\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eStatistical Analyses, Effect Sizes, and Model Validation\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe complete results of all statistical tests are summarized in \u003cstrong\u003eTable 2\u003c/strong\u003e. The Kruskal-Wallis test demonstrated significant overall group differences for total score (\u0026chi;\u0026sup2; = 8.48, p = 0.0144), bone formation (\u0026chi;\u0026sup2; = 8.99, p = 0.0112), and distal union (\u0026chi;\u0026sup2; = 10.73, p = 0.0047). Proximal union did not reach significance (p = 0.1067), while remodeling approached significance (p = 0.0563). Dunn\u0026apos;s post-hoc analysis with Bonferroni correction identified PLA+NK-75 as significantly superior to control (Z = \u0026minus;2.870, p = 0.0062), while PLA did not differ significantly from control (p = 0.7531). Mann-Whitney U testing corroborated these findings (PLA+NK-75 vs. control: U = 28.0, p = 0.0044). The Jonckheere-Terpstra test confirmed a significant ordered trend in the hypothesized direction \u0026mdash; CONTROL \u0026lt; PLA \u0026lt; PLA+NK-75 (JT = 207, p = 0.0021) \u0026mdash; validating the biological rationale that nanoclay augmentation of PLA provides additive benefit.\u003c/p\u003e\n\u003cp\u003eSensitivity analyses uniformly confirmed the primary findings. The linear mixed model, accounting for repeated measures within animals, estimated a PLA+NK-75 effect of +3.58 points over control (95% CI: 2.55\u0026ndash;4.61, t = 6.82) after adjusting for time. Each additional week contributed 0.385 points (95% CI: 0.31\u0026ndash;0.47, t = 9.48). GEE analysis with exchangeable correlation structure yielded consistent results (PLA+NK-75: Wald \u0026chi;\u0026sup2; = 64.02, p \u0026lt; 0.001). Ordinal logistic regression demonstrated dramatically increased odds of achieving higher healing categories in PLA+NK-75 (OR = 673.24, p \u0026lt; 0.001), while Poisson regression yielded a rate ratio of 1.91 (95% CI: 1.35\u0026ndash;2.74, p \u0026lt; 0.001), indicating 91% higher expected healing scores. Permutation testing with 10,000 iterations produced an exact p-value of 0.008, more stringent than the asymptotic Kruskal-Wallis p-value. Across all nine analytical methods, PLA+NK-75 versus control remained significant at p \u0026lt; 0.01, demonstrating exceptional robustness.\u003c/p\u003e\n\u003cp\u003eEffect size measures and power analysis are presented in \u003cstrong\u003eTable 3\u003c/strong\u003e. All metrics consistently indicated large treatment effects. Cliff\u0026apos;s delta of 0.661 signifies that in 66.1% of all possible pairings, a PLA+NK-75 observation exceeds a control observation. Hedges\u0026apos; g of 1.472 and Glass\u0026apos;s \u0026Delta; of 1.377 both substantially exceed the conventional threshold of 0.8 for large effects. At the global level, eta-squared of 0.323 indicates that group membership explains 32.3% of total variance in healing scores. The common language effect size translates to an 83.0% probability of PLA+NK-75 superiority over control \u0026mdash; approximately 5:1 odds. Post-hoc power analysis confirmed achieved power of 85.5%, exceeding the 80% adequacy threshold. Bootstrap 95% confidence intervals for the PLA+NK-75 median (6.0\u0026ndash;9.0) showed no overlap with the control median CI (1.0\u0026ndash;6.0), providing distribution-free confirmation of group separation.\u003c/p\u003e\n\u003cp\u003eModel comparison using information criteria identified the Group \u0026times; Time interaction model as optimal (AIC = 94.4, R\u0026sup2; = 0.880), explaining 88% of variance \u0026mdash; a substantial improvement over Group alone (R\u0026sup2; = 0.323) or Time alone (R\u0026sup2; = 0.454). PCA of the four Lane-Sandhu components revealed a dominant first principal component explaining 73.7% of variance, with all components loading positively (range: 0.352\u0026ndash;0.556), representing an overall \u0026quot;healing factor.\u0026quot; Internal consistency of the Lane-Sandhu scale was good (Cronbach\u0026apos;s \u0026alpha; = 0.866), supporting the reliability of the scoring instrument. Linear discriminant analysis achieved 73.3% classification accuracy in predicting group membership from component scores, with misclassifications occurring primarily between adjacent groups, consistent with the ordered treatment effect (\u003cstrong\u003eFigure 3\u003c/strong\u003e).\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eTable 1. Descriptive Statistics and Lane-Sandhu Component Scores by Group\u003c/strong\u003e\u003c/p\u003e\n\u003ctable border=\"1\" cellspacing=\"0\" cellpadding=\"0\" width=\"100%\"\u003e\n \u003ctbody\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\" style=\"width: 29px;\"\u003e\n \u003cp\u003e\u003cstrong\u003eParameter\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 18px;\"\u003e\n \u003cp\u003e\u003cstrong\u003eControl (n=11)\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 14px;\"\u003e\n \u003cp\u003e\u003cstrong\u003ePLA (n=4)\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 24px;\"\u003e\n \u003cp\u003e\u003cstrong\u003ePLA+NK-75 (n=15)\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 12px;\"\u003e\n \u003cp\u003e\u003cstrong\u003ep-valueᵃ\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\" style=\"width: 29px;\"\u003e\n \u003cp\u003e\u003cstrong\u003eTotal Score\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 18px;\"\u003e\u003cbr\u003e\u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 14px;\"\u003e\u003cbr\u003e\u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 24px;\"\u003e\u003cbr\u003e\u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 12px;\"\u003e\n \u003cp\u003e\u003cstrong\u003e0.0144*\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\" style=\"width: 29px;\"\u003e\n \u003cp\u003e\u003cstrong\u003eMean \u0026plusmn; SD\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 18px;\"\u003e\n \u003cp\u003e4.00 \u0026plusmn; 2.32\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 14px;\"\u003e\n \u003cp\u003e4.75 \u0026plusmn; 3.59\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 24px;\"\u003e\n \u003cp\u003e7.20 \u0026plusmn; 1.93\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 12px;\"\u003e\u003cbr\u003e\u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\" style=\"width: 29px;\"\u003e\n \u003cp\u003e\u003cstrong\u003eMedian (IQR)\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 18px;\"\u003e\n \u003cp\u003e4.0 (4.00)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 14px;\"\u003e\n \u003cp\u003e5.5 (4.25)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 24px;\"\u003e\n \u003cp\u003e7.0 (3.00)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 12px;\"\u003e\u003cbr\u003e\u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\" style=\"width: 29px;\"\u003e\n \u003cp\u003e\u003cstrong\u003eRange\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 18px;\"\u003e\n \u003cp\u003e1\u0026ndash;7\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 14px;\"\u003e\n \u003cp\u003e0\u0026ndash;8\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 24px;\"\u003e\n \u003cp\u003e4\u0026ndash;10\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 12px;\"\u003e\u003cbr\u003e\u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\" style=\"width: 29px;\"\u003e\n \u003cp\u003e\u003cstrong\u003eBone Formation (0\u0026ndash;4)\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 18px;\"\u003e\n \u003cp\u003e2.27 \u0026plusmn; 1.01\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 14px;\"\u003e\n \u003cp\u003e2.25 \u0026plusmn; 1.71\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 24px;\"\u003e\n \u003cp\u003e3.47 \u0026plusmn; 0.64\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 12px;\"\u003e\n \u003cp\u003e\u003cstrong\u003e0.0112*\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\" style=\"width: 29px;\"\u003e\n \u003cp\u003e\u003cstrong\u003eProximal Union (0\u0026ndash;2)\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 18px;\"\u003e\n \u003cp\u003e1.18 \u0026plusmn; 0.87\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 14px;\"\u003e\n \u003cp\u003e1.25 \u0026plusmn; 0.96\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 24px;\"\u003e\n \u003cp\u003e1.80 \u0026plusmn; 0.41\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 12px;\"\u003e\n \u003cp\u003e0.1067\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\" style=\"width: 29px;\"\u003e\n \u003cp\u003e\u003cstrong\u003eDistal Union (0\u0026ndash;2)\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 18px;\"\u003e\n \u003cp\u003e0.55 \u0026plusmn; 0.52\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 14px;\"\u003e\n \u003cp\u003e1.25 \u0026plusmn; 0.96\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 24px;\"\u003e\n \u003cp\u003e1.47 \u0026plusmn; 0.52\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 12px;\"\u003e\n \u003cp\u003e\u003cstrong\u003e0.0047**\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\" style=\"width: 29px;\"\u003e\n \u003cp\u003e\u003cstrong\u003eRemodeling (0\u0026ndash;2)\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 18px;\"\u003e\n \u003cp\u003e0.00 \u0026plusmn; 0.00\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 14px;\"\u003e\n \u003cp\u003e0.00 \u0026plusmn; 0.00\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 24px;\"\u003e\n \u003cp\u003e0.47 \u0026plusmn; 0.74\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 12px;\"\u003e\n \u003cp\u003e0.0563\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\" style=\"width: 29px;\"\u003e\n \u003cp\u003e\u003cstrong\u003eScore by Time Pointᵇ\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 18px;\"\u003e\u003cbr\u003e\u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 14px;\"\u003e\u003cbr\u003e\u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 24px;\"\u003e\u003cbr\u003e\u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 12px;\"\u003e\u003cbr\u003e\u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\" style=\"width: 29px;\"\u003e\n \u003cp\u003e\u003cstrong\u003eWeek 4\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 18px;\"\u003e\n \u003cp\u003e1.0\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 14px;\"\u003e\n \u003cp\u003e0.0\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 24px;\"\u003e\n \u003cp\u003e5.2\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 12px;\"\u003e\u003cbr\u003e\u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\" style=\"width: 29px;\"\u003e\n \u003cp\u003e\u003cstrong\u003eWeek 8\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 18px;\"\u003e\n \u003cp\u003e4.0\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 14px;\"\u003e\n \u003cp\u003e4.0\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 24px;\"\u003e\n \u003cp\u003e6.75\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 12px;\"\u003e\u003cbr\u003e\u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\" style=\"width: 29px;\"\u003e\n \u003cp\u003e\u003cstrong\u003eWeek 12\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 18px;\"\u003e\n \u003cp\u003e5.0\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 14px;\"\u003e\n \u003cp\u003e7.0\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 24px;\"\u003e\n \u003cp\u003e9.0\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 12px;\"\u003e\u003cbr\u003e\u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\" style=\"width: 29px;\"\u003e\n \u003cp\u003e\u003cstrong\u003eWeek 16\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 18px;\"\u003e\n \u003cp\u003e6.0\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 14px;\"\u003e\n \u003cp\u003e8.0\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 24px;\"\u003e\n \u003cp\u003e9.0\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 12px;\"\u003e\u003cbr\u003e\u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\" style=\"width: 29px;\"\u003e\n \u003cp\u003e\u003cstrong\u003eWeek 20\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 18px;\"\u003e\n \u003cp\u003e7.0\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 14px;\"\u003e\n \u003cp\u003eNA\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 24px;\"\u003e\n \u003cp\u003e10.0\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 12px;\"\u003e\u003cbr\u003e\u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\" style=\"width: 29px;\"\u003e\n \u003cp\u003e\u003cstrong\u003eHealing Category, n (%)\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 18px;\"\u003e\u003cbr\u003e\u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 14px;\"\u003e\u003cbr\u003e\u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 24px;\"\u003e\u003cbr\u003e\u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 12px;\"\u003e\n \u003cp\u003e\u003cstrong\u003e0.0025\u003c/strong\u003eᶜ\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\" style=\"width: 29px;\"\u003e\n \u003cp\u003e\u003cstrong\u003eNo Healing / Poor (0\u0026ndash;3)\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 18px;\"\u003e\n \u003cp\u003e4 (36.4%)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 14px;\"\u003e\n \u003cp\u003e1 (25.0%)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 24px;\"\u003e\n \u003cp\u003e1 (6.7%)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 12px;\"\u003e\u003cbr\u003e\u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\" style=\"width: 29px;\"\u003e\n \u003cp\u003e\u003cstrong\u003eFair (4\u0026ndash;5)\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 18px;\"\u003e\n \u003cp\u003e3 (27.3%)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 14px;\"\u003e\n \u003cp\u003e1 (25.0%)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 24px;\"\u003e\n \u003cp\u003e1 (6.7%)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 12px;\"\u003e\u003cbr\u003e\u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\" style=\"width: 29px;\"\u003e\n \u003cp\u003e\u003cstrong\u003eGood (6\u0026ndash;7)\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 18px;\"\u003e\n \u003cp\u003e4 (36.4%)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 14px;\"\u003e\n \u003cp\u003e1 (25.0%)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 24px;\"\u003e\n \u003cp\u003e6 (40.0%)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 12px;\"\u003e\u003cbr\u003e\u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\" style=\"width: 29px;\"\u003e\n \u003cp\u003e\u003cstrong\u003eExcellent (8\u0026ndash;10)\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 18px;\"\u003e\n \u003cp\u003e0 (0%)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 14px;\"\u003e\n \u003cp\u003e1 (25.0%)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 24px;\"\u003e\n \u003cp\u003e7 (46.7%)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 12px;\"\u003e\u003cbr\u003e\u003c/td\u003e\n \u003c/tr\u003e\n \u003c/tbody\u003e\n\u003c/table\u003e\n\u003cp\u003eᵃKruskal-Wallis test. ᵇMean total scores. ᶜFisher\u0026apos;s Exact Test. *p \u0026lt; 0.05; **p \u0026lt; 0.01. NA = not available (no PLA observations at week 20).\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eTable 2. Summary of Statistical Significance Across All Analytical Methods\u003c/strong\u003e\u003c/p\u003e\n\u003ctable border=\"1\" cellspacing=\"0\" cellpadding=\"0\" width=\"100%\"\u003e\n \u003ctbody\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\" style=\"width: 28px;\"\u003e\n \u003cp\u003e\u003cstrong\u003eAnalysis Method\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 15px;\"\u003e\n \u003cp\u003e\u003cstrong\u003eTest Statistic\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 22px;\"\u003e\n \u003cp\u003e\u003cstrong\u003ePLA+NK-75 vs Control\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 15px;\"\u003e\n \u003cp\u003e\u003cstrong\u003ePLA vs Control\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 17px;\"\u003e\n \u003cp\u003e\u003cstrong\u003eOverall p-value\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\" style=\"width: 28px;\"\u003e\n \u003cp\u003e\u003cstrong\u003ePrimary Non-parametric\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 15px;\"\u003e\u003cbr\u003e\u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 22px;\"\u003e\u003cbr\u003e\u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 15px;\"\u003e\u003cbr\u003e\u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 17px;\"\u003e\u003cbr\u003e\u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\" style=\"width: 28px;\"\u003e\n \u003cp\u003e\u003cstrong\u003eKruskal-Wallis\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 15px;\"\u003e\n \u003cp\u003e\u0026chi;\u0026sup2; = 8.48\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 22px;\"\u003e\n \u003cp\u003e\u0026mdash;\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 15px;\"\u003e\n \u003cp\u003e\u0026mdash;\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 17px;\"\u003e\n \u003cp\u003e0.0144*\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\" style=\"width: 28px;\"\u003e\n \u003cp\u003e\u003cstrong\u003eDunn\u0026apos;s Post-hoc (Bonferroni)\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 15px;\"\u003e\n \u003cp\u003eZ = \u0026minus;2.870\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 22px;\"\u003e\n \u003cp\u003e\u003cstrong\u003e0.0062**\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 15px;\"\u003e\n \u003cp\u003e0.7531\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 17px;\"\u003e\n \u003cp\u003e\u0026mdash;\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\" style=\"width: 28px;\"\u003e\n \u003cp\u003e\u003cstrong\u003eMann-Whitney U\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 15px;\"\u003e\n \u003cp\u003eU = 28.0\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 22px;\"\u003e\n \u003cp\u003e\u003cstrong\u003e0.0044**\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 15px;\"\u003e\n \u003cp\u003e0.5507\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 17px;\"\u003e\n \u003cp\u003e\u0026mdash;\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\" style=\"width: 28px;\"\u003e\n \u003cp\u003e\u003cstrong\u003eJonckheere-Terpstra\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 15px;\"\u003e\n \u003cp\u003eJT = 207\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 22px;\"\u003e\n \u003cp\u003e\u0026mdash;\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 15px;\"\u003e\n \u003cp\u003e\u0026mdash;\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 17px;\"\u003e\n \u003cp\u003e0.0021**\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\" style=\"width: 28px;\"\u003e\n \u003cp\u003e\u003cstrong\u003ePermutation Test (10,000 iter.)\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 15px;\"\u003e\n \u003cp\u003e\u0026mdash;\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 22px;\"\u003e\n \u003cp\u003e\u0026mdash;\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 15px;\"\u003e\n \u003cp\u003e\u0026mdash;\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 17px;\"\u003e\n \u003cp\u003e0.0080**\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\" style=\"width: 28px;\"\u003e\n \u003cp\u003e\u003cstrong\u003eFisher\u0026apos;s Exact (categories)\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 15px;\"\u003e\n \u003cp\u003e\u0026mdash;\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 22px;\"\u003e\n \u003cp\u003e\u0026mdash;\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 15px;\"\u003e\n \u003cp\u003e\u0026mdash;\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 17px;\"\u003e\n \u003cp\u003e0.0025**\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\" style=\"width: 28px;\"\u003e\n \u003cp\u003e\u003cstrong\u003eSensitivity / Regression Models\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 15px;\"\u003e\u003cbr\u003e\u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 22px;\"\u003e\u003cbr\u003e\u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 15px;\"\u003e\u003cbr\u003e\u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 17px;\"\u003e\u003cbr\u003e\u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\" style=\"width: 28px;\"\u003e\n \u003cp\u003e\u003cstrong\u003eLinear Mixed Model\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 15px;\"\u003e\n \u003cp\u003et = 6.82\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 22px;\"\u003e\n \u003cp\u003e\u003cstrong\u003e\u0026lt;0.001***\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 15px;\"\u003e\n \u003cp\u003e0.273\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 17px;\"\u003e\n \u003cp\u003e\u0026mdash;\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\" style=\"width: 28px;\"\u003e\n \u003cp\u003e\u003cstrong\u003eGEE (exchangeable)\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 15px;\"\u003e\n \u003cp\u003eWald \u0026chi;\u0026sup2; = 64.02\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 22px;\"\u003e\n \u003cp\u003e\u003cstrong\u003e\u0026lt;0.001***\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 15px;\"\u003e\n \u003cp\u003e0.005**\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 17px;\"\u003e\n \u003cp\u003e\u0026mdash;\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\" style=\"width: 28px;\"\u003e\n \u003cp\u003e\u003cstrong\u003eOrdinal Logistic Regression\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 15px;\"\u003e\n \u003cp\u003eOR = 673.24\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 22px;\"\u003e\n \u003cp\u003e\u003cstrong\u003e\u0026lt;0.001***\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 15px;\"\u003e\n \u003cp\u003e0.304\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 17px;\"\u003e\n \u003cp\u003e\u0026mdash;\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\" style=\"width: 28px;\"\u003e\n \u003cp\u003e\u003cstrong\u003ePoisson Regression\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 15px;\"\u003e\n \u003cp\u003eRR = 1.91\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 22px;\"\u003e\n \u003cp\u003e\u003cstrong\u003e\u0026lt;0.001***\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 15px;\"\u003e\n \u003cp\u003e0.465\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 17px;\"\u003e\n \u003cp\u003e\u0026mdash;\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\" style=\"width: 28px;\"\u003e\n \u003cp\u003e\u003cstrong\u003eCorrelation Analyses\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 15px;\"\u003e\u003cbr\u003e\u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 22px;\"\u003e\u003cbr\u003e\u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 15px;\"\u003e\u003cbr\u003e\u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 17px;\"\u003e\u003cbr\u003e\u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\" style=\"width: 28px;\"\u003e\n \u003cp\u003e\u003cstrong\u003eSpearman \u0026rho; (Time vs Score)\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 15px;\"\u003e\n \u003cp\u003e\u0026rho; = 0.705\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 22px;\"\u003e\n \u003cp\u003e\u0026mdash;\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 15px;\"\u003e\n \u003cp\u003e\u0026mdash;\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 17px;\"\u003e\n \u003cp\u003e\u0026lt;0.0001***\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\" style=\"width: 28px;\"\u003e\n \u003cp\u003e\u003cstrong\u003eLinear Trend (ordered groups)\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 15px;\"\u003e\n \u003cp\u003er = 0.539\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 22px;\"\u003e\n \u003cp\u003e\u0026mdash;\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 15px;\"\u003e\n \u003cp\u003e\u0026mdash;\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 17px;\"\u003e\n \u003cp\u003e0.0021**\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003c/tbody\u003e\n\u003c/table\u003e\n\u003cp\u003e*p \u0026lt; 0.05; **p \u0026lt; 0.01; ***p \u0026lt; 0.001. GEE = Generalized Estimating Equations; RR = Rate Ratio; OR = Odds Ratio.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eTable 3. Effect Size Measures and Statistical Power\u003c/strong\u003e\u003c/p\u003e\n\u003ctable border=\"1\" cellspacing=\"0\" cellpadding=\"0\" width=\"100%\"\u003e\n \u003ctbody\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\" style=\"width: 31px;\"\u003e\n \u003cp\u003e\u003cstrong\u003ePairwise Effect Sizes\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 22px;\"\u003e\n \u003cp\u003e\u003cstrong\u003ePLA+NK-75 vs Control\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 29px;\"\u003e\n \u003cp\u003e\u003cstrong\u003ePLA+NK-75 vs PLA\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 16px;\"\u003e\n \u003cp\u003e\u003cstrong\u003ePLA vs Control\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\" style=\"width: 31px;\"\u003e\n \u003cp\u003e\u003cstrong\u003eCliff\u0026apos;s Delta\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 22px;\"\u003e\n \u003cp\u003e0.661 (Large)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 29px;\"\u003e\n \u003cp\u003e0.433 (Medium)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 16px;\"\u003e\n \u003cp\u003e0.227 (Small)\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\" style=\"width: 31px;\"\u003e\n \u003cp\u003e\u003cstrong\u003eHedges\u0026apos; g\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 22px;\"\u003e\n \u003cp\u003e1.472 (Large)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 29px;\"\u003e\n \u003cp\u003e1.011 (Large)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 16px;\"\u003e\n \u003cp\u003e0.264 (Small)\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\" style=\"width: 31px;\"\u003e\n \u003cp\u003e\u003cstrong\u003eGlass\u0026apos;s \u0026Delta;\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 22px;\"\u003e\n \u003cp\u003e1.377 (Large)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 29px;\"\u003e\n \u003cp\u003e\u0026mdash;\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 16px;\"\u003e\n \u003cp\u003e0.323 (Small)\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\" style=\"width: 31px;\"\u003e\n \u003cp\u003e\u003cstrong\u003eProbability of Superiority\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 22px;\"\u003e\n \u003cp\u003e83.0%\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 29px;\"\u003e\n \u003cp\u003e\u0026mdash;\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 16px;\"\u003e\n \u003cp\u003e61.4%\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\" style=\"width: 31px;\"\u003e\n \u003cp\u003e\u003cstrong\u003eGlobal Effect Sizes\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 22px;\"\u003e\n \u003cp\u003e\u003cstrong\u003eValue\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 29px;\"\u003e\n \u003cp\u003e\u003cstrong\u003eInterpretation\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 16px;\"\u003e\u003cbr\u003e\u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\" style=\"width: 31px;\"\u003e\n \u003cp\u003e\u003cstrong\u003eEpsilon-squared (\u0026epsilon;\u0026sup2;)\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 22px;\"\u003e\n \u003cp\u003e0.292\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 29px;\"\u003e\n \u003cp\u003eLarge\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 16px;\"\u003e\u003cbr\u003e\u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\" style=\"width: 31px;\"\u003e\n \u003cp\u003e\u003cstrong\u003eEta-squared (\u0026eta;\u0026sup2;)\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 22px;\"\u003e\n \u003cp\u003e0.323\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 29px;\"\u003e\n \u003cp\u003eLarge (32.3% variance explained)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 16px;\"\u003e\u003cbr\u003e\u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\" style=\"width: 31px;\"\u003e\n \u003cp\u003e\u003cstrong\u003eCohen\u0026apos;s f\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 22px;\"\u003e\n \u003cp\u003e0.643\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 29px;\"\u003e\n \u003cp\u003eLarge\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 16px;\"\u003e\u003cbr\u003e\u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\" style=\"width: 31px;\"\u003e\n \u003cp\u003e\u003cstrong\u003ePower and Model Fit\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 22px;\"\u003e\n \u003cp\u003e\u003cstrong\u003eValue\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 29px;\"\u003e\n \u003cp\u003e\u003cstrong\u003eInterpretation\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 16px;\"\u003e\u003cbr\u003e\u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\" style=\"width: 31px;\"\u003e\n \u003cp\u003e\u003cstrong\u003eAchieved Power\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 22px;\"\u003e\n \u003cp\u003e85.5%\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 29px;\"\u003e\n \u003cp\u003eAdequate (\u0026gt;80%)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 16px;\"\u003e\u003cbr\u003e\u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\" style=\"width: 31px;\"\u003e\n \u003cp\u003e\u003cstrong\u003eBest Model R\u0026sup2; (Group \u0026times; Time)\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 22px;\"\u003e\n \u003cp\u003e0.880\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 29px;\"\u003e\n \u003cp\u003e88% variance explained\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 16px;\"\u003e\u003cbr\u003e\u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\" style=\"width: 31px;\"\u003e\n \u003cp\u003e\u003cstrong\u003eCronbach\u0026apos;s \u0026alpha; (Lane-Sandhu)\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 22px;\"\u003e\n \u003cp\u003e0.866\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 29px;\"\u003e\n \u003cp\u003eGood internal consistency\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 16px;\"\u003e\u003cbr\u003e\u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\" style=\"width: 31px;\"\u003e\n \u003cp\u003e\u003cstrong\u003eBootstrap 95% CI: PLA+NK-75 median\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 22px;\"\u003e\n \u003cp\u003e6.0\u0026ndash;9.0\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 29px;\"\u003e\n \u003cp\u003eNon-overlapping with Control CI\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 16px;\"\u003e\u003cbr\u003e\u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\" style=\"width: 31px;\"\u003e\n \u003cp\u003e\u003cstrong\u003eBootstrap 95% CI: Control median\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 22px;\"\u003e\n \u003cp\u003e1.0\u0026ndash;6.0\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 29px;\"\u003e\n \u003cp\u003e\u0026mdash;\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 16px;\"\u003e\u003cbr\u003e\u003c/td\u003e\n \u003c/tr\u003e\n \u003c/tbody\u003e\n\u003c/table\u003e\n\u003cp\u003e\u003cbr\u003e\u003c/p\u003e"},{"header":"DISCUSSION","content":"\u003cp\u003eThe present study demonstrates that a PLA/NK-75 nanoclay biodegradable composite scaffold significantly enhances radiological bone healing in a rabbit critical-size ulnar defect model, with large effect sizes, accelerated milestone achievement, and exclusive remodeling activity. These findings contribute meaningfully to the growing body of evidence supporting nanoclay-augmented scaffolds for orthopaedic bone regeneration.\u003c/p\u003e\n\u003cp\u003eThe superior bone formation observed in the PLA+NK-75 group (mean bone formation score 3.47 ± 0.64 versus 2.27 ± 1.01 for controls) aligns with the findings of Lopresti et al. (2021), who demonstrated that incorporation of nanoclay into electrospun PLA nanofibers significantly enhanced pre-osteoblastic cell proliferation and improved surface wettability and mechanical properties compared to neat PLA scaffolds [15]. Our in vivo results thus provide biological confirmation of the in vitro advantages reported for PLA/nanoclay systems. Similarly, Sinha Ray (2012) reported in a comprehensive review that adding approximately 5 vol% clay to PLA improved storage modulus, tensile strength, and surface characteristics critical for tissue engineering, findings consistent with the 5 wt% nanoclay concentration employed in our composite [16].\u003c/p\u003e\n\u003cp\u003eThe observation that remodeling activity was exclusively confined to the PLA+NK-75 group is particularly noteworthy. Neither the PLA-only nor the control group demonstrated any remodeling throughout the 20-week observation period, indicating that nanoclay augmentation was essential for healing to progress to its final maturation phase. This finding is consistent with the work of Ke et al. (2023), who showed that montmorillonite incorporation into keratin hydrogels dramatically enhanced osteogenic differentiation via the BMP-2/p-SMAD 1/5/8/RUNX2 signaling pathway and promoted superior bone regeneration in a rat cranial defect model [17]. The bioactive ions (silicon, magnesium, aluminium) released from montmorillonite-based nanoclays are known to stimulate osteoblastic activity and matrix mineralization, which may explain the advanced healing maturity observed exclusively in the nanoclay-containing group.\u003c/p\u003e\n\u003cp\u003eHu et al. (2020) provided important mechanistic insight by demonstrating that nanoclay's primary contribution to bone regeneration is through drug binding and sustained release of osteogenic growth factors rather than intrinsic osteoinductivity alone [18]. Although our scaffold was not loaded with exogenous growth factors, the sustained release of endogenous bioactive ions from the NK-75 montmorillonite may have functioned through an analogous mechanism of prolonged local bioactivity, supporting the progressive healing trajectory observed in the PLA+NK-75 group from week 4 through week 20 without plateau.\u003c/p\u003e\n\u003cp\u003eThe significant ordered trend (CONTROL \u0026lt; PLA \u0026lt; PLA+NK-75; Jonckheere-Terpstra p = 0.0021) validates the additive contribution of nanoclay to the polymer scaffold. This dose-response pattern is supported by Huang et al. (2019), who demonstrated that halloysite nanotube (a naturally occurring aluminosilicate nanoclay) incorporation into GelMA hydrogels significantly upregulated osteogenic gene expression and enhanced calvarial bone regeneration in rats compared to neat polymer scaffolds [19]. Li et al. (2024) further corroborated the osteogenic superiority of nanoclay-containing scaffolds, reporting that GelMA-nanoclay hydrogels enhanced osteogenesis, angiogenesis, and immunomodulation in bone defects [20].\u003c/p\u003e\n\u003cp\u003eOur use of the Lane-Sandhu scoring system is consistent with the methodology employed by Basaran et al. (2022), who used a modified Lane-Sandhu system to evaluate bone healing in a rabbit ulnar segmental defect model [21]. However, whereas their biocomposite scaffold without mesenchymal stem cells achieved satisfactory regeneration by 6 weeks, our PLA+NK-75 composite achieved good healing (score ≥6) as early as week 4, suggesting that nanoclay-enhanced scaffolds may accelerate early-phase healing even without exogenous cellular supplementation.\u003c/p\u003e\n\u003cp\u003eThe 83% probability of superiority of PLA+NK-75 over controls and the 88% variance explained by the Group × Time interaction model underscore both the magnitude and reliability of the treatment effect. Salehi et al. (2024) similarly reported that PLA-based nanocomposite scaffolds incorporating bredigite nanoparticles achieved near-complete calvarial bone recovery in rats within 8 weeks [22], supporting the principle that bioactive nanofiller incorporation into PLA matrices substantially amplifies regenerative capacity. Furthermore, Zhang et al. (2021) demonstrated that even 1% w/v nanosilicate incorporation into silk fibroin hydrogels was sufficient to enhance dual-lineage differentiation and promote osteochondral regeneration in rabbit defects [23], suggesting that nanoclays exert potent biological effects at relatively low concentrations.\u003c/p\u003e\n\u003cp\u003eSeveral limitations of this study warrant consideration. The unequal group sizes, particularly the small PLA-only group (n = 4), limit the statistical power for PLA versus control comparisons, although the primary PLA+NK-75 versus control comparison achieved adequate power (85.5%). The study relied exclusively on radiological assessment using the Lane-Sandhu system without quantitative histomorphometry or micro-CT analysis. The rabbit ulnar model, while well-established, does not replicate the biomechanical loading environment of human long bones. Future studies should incorporate histomorphometric quantification, micro-CT volumetric analysis, and biomechanical testing, along with dose-optimization of nanoclay concentration and evaluation in larger animal models to further validate these findings prior to clinical translation.\u003c/p\u003e"},{"header":"CONCLUSION","content":"\u003cp\u003eThe PLA/NK-75 biodegradable nanoclay composite scaffold demonstrated statistically significant and clinically meaningful superiority over both PLA alone and untreated empty defect controls in promoting radiological bone healing in a rabbit critical-size ulnar defect model. The composite achieved the highest Lane-Sandhu scores at every time point, with large effect sizes, strong probability of superiority over controls, and markedly accelerated healing milestones. Remodeling, the final phase of bone regeneration, was exclusively observed in the PLA+NK-75 group, indicating the composite supports healing to a degree of maturity unattainable by PLA alone or natural repair. A significant ordered trend validated the hypothesis that nanoclay augmentation provides additive benefit to the biodegradable polymer scaffold. These results were robust across multiple independent statistical methods, strengthening causal inference. The findings support PLA/NK-75 nanoclay composite as a promising, cost-effective scaffold material for orthopaedic bone regeneration that warrants further preclinical investigation with histomorphometric, micro-CT, and biomechanical endpoints, dose-optimization studies, and eventual translation toward larger animal models.\u003c/p\u003e\n\u003cp\u003e\u003cbr\u003e\u003c/p\u003e"},{"header":"Abbreviations","content":"\u003cdiv class=\"DefinitionList\"\u003e \u003cdiv class=\"DefinitionListEntry\"\u003e \u003cdiv class=\"Term\"\u003eAIC\u003c/div\u003e \u003cdiv class=\"Description\"\u003e \u003cp\u003eAkaike information criterion\u003c/p\u003e \u003c/div\u003e \u003c/div\u003e \u003cdiv class=\"DefinitionListEntry\"\u003e \u003cdiv class=\"Term\"\u003eARRIVE\u003c/div\u003e \u003cdiv class=\"Description\"\u003e \u003cp\u003eAnimal Research:Reporting of In Vivo Experiments\u003c/p\u003e \u003c/div\u003e \u003c/div\u003e \u003cdiv class=\"DefinitionListEntry\"\u003e \u003cdiv class=\"Term\"\u003eBIC\u003c/div\u003e \u003cdiv class=\"Description\"\u003e \u003cp\u003eBayesian information criterion\u003c/p\u003e \u003c/div\u003e \u003c/div\u003e \u003cdiv class=\"DefinitionListEntry\"\u003e \u003cdiv class=\"Term\"\u003eBMP-2\u003c/div\u003e \u003cdiv class=\"Description\"\u003e \u003cp\u003eBone morphogenetic protein 2\u003c/p\u003e \u003c/div\u003e \u003c/div\u003e \u003cdiv class=\"DefinitionListEntry\"\u003e \u003cdiv class=\"Term\"\u003eCI\u003c/div\u003e \u003cdiv class=\"Description\"\u003e \u003cp\u003eConfidence interval\u003c/p\u003e \u003c/div\u003e \u003c/div\u003e \u003cdiv class=\"DefinitionListEntry\"\u003e \u003cdiv class=\"Term\"\u003eCPCSEA\u003c/div\u003e \u003cdiv class=\"Description\"\u003e \u003cp\u003eCommittee for the Purpose of Control and Supervision of Experiments on Animals\u003c/p\u003e \u003c/div\u003e \u003c/div\u003e \u003cdiv class=\"DefinitionListEntry\"\u003e \u003cdiv class=\"Term\"\u003eEDAX\u003c/div\u003e \u003cdiv class=\"Description\"\u003e \u003cp\u003eEnergy-dispersive X-ray analysis\u003c/p\u003e \u003c/div\u003e \u003c/div\u003e \u003cdiv class=\"DefinitionListEntry\"\u003e \u003cdiv class=\"Term\"\u003eFDA\u003c/div\u003e \u003cdiv class=\"Description\"\u003e \u003cp\u003eFood and Drug Administration\u003c/p\u003e \u003c/div\u003e \u003c/div\u003e \u003cdiv class=\"DefinitionListEntry\"\u003e \u003cdiv class=\"Term\"\u003eGEE\u003c/div\u003e \u003cdiv class=\"Description\"\u003e \u003cp\u003eGeneralised estimating equations\u003c/p\u003e \u003c/div\u003e \u003c/div\u003e \u003cdiv class=\"DefinitionListEntry\"\u003e \u003cdiv class=\"Term\"\u003eGelMA\u003c/div\u003e \u003cdiv class=\"Description\"\u003e \u003cp\u003eGelatin methacryloyl\u003c/p\u003e \u003c/div\u003e \u003c/div\u003e \u003cdiv class=\"DefinitionListEntry\"\u003e \u003cdiv class=\"Term\"\u003eH\u0026amp;E\u003c/div\u003e \u003cdiv class=\"Description\"\u003e \u003cp\u003eHaematoxylin and eosin\u003c/p\u003e \u003c/div\u003e \u003c/div\u003e \u003cdiv class=\"DefinitionListEntry\"\u003e \u003cdiv class=\"Term\"\u003eIAEC\u003c/div\u003e \u003cdiv class=\"Description\"\u003e \u003cp\u003eInstitutional Animal Ethics Committee\u003c/p\u003e \u003c/div\u003e \u003c/div\u003e \u003cdiv class=\"DefinitionListEntry\"\u003e \u003cdiv class=\"Term\"\u003eIQR\u003c/div\u003e \u003cdiv class=\"Description\"\u003e \u003cp\u003eInterquartile range\u003c/p\u003e \u003c/div\u003e \u003c/div\u003e \u003cdiv class=\"DefinitionListEntry\"\u003e \u003cdiv class=\"Term\"\u003eNK-75\u003c/div\u003e \u003cdiv class=\"Description\"\u003e \u003cp\u003eNanoclay (organically modified montmorillonite)\u003c/p\u003e \u003c/div\u003e \u003c/div\u003e \u003cdiv class=\"DefinitionListEntry\"\u003e \u003cdiv class=\"Term\"\u003eOR\u003c/div\u003e \u003cdiv class=\"Description\"\u003e \u003cp\u003eOdds ratio\u003c/p\u003e \u003c/div\u003e \u003c/div\u003e \u003cdiv class=\"DefinitionListEntry\"\u003e \u003cdiv class=\"Term\"\u003ePCA\u003c/div\u003e \u003cdiv class=\"Description\"\u003e \u003cp\u003ePrincipal component analysis\u003c/p\u003e \u003c/div\u003e \u003c/div\u003e \u003cdiv class=\"DefinitionListEntry\"\u003e \u003cdiv class=\"Term\"\u003ePLA\u003c/div\u003e \u003cdiv class=\"Description\"\u003e \u003cp\u003ePolylactic acid\u003c/p\u003e \u003c/div\u003e \u003c/div\u003e \u003cdiv class=\"DefinitionListEntry\"\u003e \u003cdiv class=\"Term\"\u003eRR\u003c/div\u003e \u003cdiv class=\"Description\"\u003e \u003cp\u003eRate ratio\u003c/p\u003e \u003c/div\u003e \u003c/div\u003e \u003cdiv class=\"DefinitionListEntry\"\u003e \u003cdiv class=\"Term\"\u003eRUNX2\u003c/div\u003e \u003cdiv class=\"Description\"\u003e \u003cp\u003eRunt-related transcription factor 2\u003c/p\u003e \u003c/div\u003e \u003c/div\u003e \u003cdiv class=\"DefinitionListEntry\"\u003e \u003cdiv class=\"Term\"\u003eSD\u003c/div\u003e \u003cdiv class=\"Description\"\u003e \u003cp\u003eStandard deviation\u003c/p\u003e \u003c/div\u003e \u003c/div\u003e \u003c/div\u003e"},{"header":"Declarations","content":"\u003cp\u003e\u003cstrong\u003eEthics approval and consent to participate\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThis study was approved by the Institutional Animal Ethics Committee (IAEC), Institute of Medical Sciences, Banaras Hindu University, Varanasi, India (Approval No. Dean/2023/IAEC/6193, dated 10.08.2023; CPCSEA Registration No. 542/GO/ReBi//S/02/CCSEA, dated 17.07.2023). All experimental procedures were conducted in accordance with the guidelines of the Committee for the Purpose of Control and Supervision of Experiments on Animals (CPCSEA), Government of India.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eConsent for publication:\u0026nbsp;\u003c/strong\u003eNot applicable.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eClinical trial registration:\u0026nbsp;\u003c/strong\u003eNot applicable.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAvailability of data and materials\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe datasets generated and/or analysed during the current study are available from the corresponding author on reasonable request.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eCompeting interests\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe authors declare that they have no competing interests.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eFunding\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThis research did not receive any specific grant from funding agencies in the public, commercial, or not-for-profit sectors.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAuthors' contributions\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eAR: Conceptualisation, Methodology, Investigation, Formal analysis, Data curation, Visualisation, Writing — original draft. AS: Conceptualisation, Methodology, Supervision, Project administration, Resources, Validation, Writing — review and editing. PM: Methodology, Resources, Supervision, Writing — review and editing. SY: Investigation, Validation, Writing — review and editing. AG: Investigation (histological evaluation and interpretation of H\u0026amp;E, Toluidine Blue O, and Von Kossa stained section, Validation, Writing — review and editing. SM: Methodology, Investigation, Resources. AnR: Investigation, Data curation, Writing — review and editing. SRN: Investigation, Data curation. SJ: Investigation, Data curation. All authors read and approved the final manuscript.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAcknowledgements:\u0026nbsp;\u003c/strong\u003eNot applicable.\u003c/p\u003e"},{"header":"References","content":"\u003col\u003e\u003cli\u003e\u003cspan\u003e Rodham PL, Giannoudis VP, Kanakaris NK, Giannoudis PV. Biological aspects to enhance fracture healing. EFORT Open Rev. 2023;8(5):264\u0026ndash;282. doi:10.1530/EOR-23-0047\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003e Pereira HF, Cengiz IF, Silva FS, Reis RL, Oliveira JM. Scaffolds and coatings for bone regeneration. J Mater Sci Mater Med. 2020;31(3):27. doi:10.1007/s10856-020-06364-y\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003e Busch A, J\u0026auml;ger M. Synthetic bone replacement substances. Orthop\u0026auml;die (Heidelb). 2022;51(12):1023\u0026ndash;1032. doi:10.1007/s00132-022-04319-5\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003e Wang Y, Wang J, Gao R, et al. Biomimetic glycopeptide hydrogel coated PCL/nHA scaffold for enhanced cranial bone regeneration via macrophage M2 polarization-induced osteo-immunomodulation. Biomaterials. 2022;285:121538. doi:10.1016/j.biomaterials.2022.121538\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003e Arif U, Haider S, Haider A, et al. Biocompatible polymers and their potential biomedical applications: a review. Curr Pharm Des. 2019;25(34):3608\u0026ndash;3619. doi:10.2174/1381612825999191011105148\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003e Alavi MS, Memarpour S, Pazhohan-Nezhad H, Salimi Asl A, Moghbeli M, Shadmanfar S, Saburi E. Applications of poly(lactic acid) in bone tissue engineering: a review article. Artif Organs. 2023;47(9):1423\u0026ndash;1430. doi:10.1111/aor.14612\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003e Tyler B, Gullotti D, Mangraviti A, Utsuki T, Brem H. Polylactic acid (PLA) controlled delivery carriers for biomedical applications. Adv Drug Deliv Rev. 2016;107:163\u0026ndash;175. doi:10.1016/j.addr.2016.06.018\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003e Mohan A, Girdhar M, Kumar R, et al. Polyhydroxybutyrate-based nanocomposites for bone tissue engineering. Pharmaceuticals (Basel). 2021;14(11):1163. doi:10.3390/ph14111163\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003e Katti KS, Jasuja H, Jaswandkar SV, Mohanty S, Katti DR. Nanoclays in medicine: a new frontier of an ancient medical practice. Mater Adv. 2022;3(20):7484\u0026ndash;7500. doi:10.1039/d2ma00528j\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003e Yao Q, Fuglsby KE, Zheng X, Sun H. Nanoclay-functionalized 3D nanofibrous scaffolds promote bone regeneration. J Mater Chem B. 2020;8(17):3842\u0026ndash;3851. doi:10.1039/c9tb02814e\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003e Zheng X, Zhang X, Wang Y, et al. Hypoxia-mimicking 3D bioglass-nanoclay scaffolds promote endogenous bone regeneration. Bioact Mater. 2021;6(10):3485\u0026ndash;3495. doi:10.1016/j.bioactmat.2021.03.011\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003e Erezuma I, Lukin I, Pimenta-Lopes C, et al. Nanoclay-reinforced HA/alginate scaffolds as cell carriers and SDF-1 delivery-platforms for bone tissue engineering. Int J Pharm. 2022;623:121895. doi:10.1016/j.ijpharm.2022.121895\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003e Kapusetti G, Misra N, Singh V, et al. Bone cement based nanohybrid as a super biomaterial for bone healing. J Mater Chem B. 2014;2(25):3984\u0026ndash;3997. doi:10.1039/C4TB00501E\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003e Senapati S, Mahanta AK, Kumar S, Maiti P. Controlled drug delivery vehicles for cancer treatment and their performance. Signal Transduct Target Ther. 2018;3:7. doi:10.1038/s41392-017-0004-3\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003e Lopresti F, Carf\u0026igrave; Pavia F, Ceraulo M, Capuana E, Brucato V, Ghersi G, et al. Physical and biological properties of electrospun poly(d,l-lactide)/nanoclay and poly(d,l-lactide)/nanosilica nanofibrous scaffold for bone tissue engineering. J Biomed Mater Res A. 2021;109(11):2120\u0026ndash;2136. doi:10.1002/jbm.a.37199\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003e Sinha Ray S. Polylactide-based bionanocomposites: a promising class of hybrid materials. Acc Chem Res. 2012;45(10):1710\u0026ndash;1720. doi:10.1021/ar3000376\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003e Ke Y, Wu J, Ye Y, Zhang X, Gu T, Wang Y, et al. Feather keratin-montmorillonite nanocomposite hydrogel promotes bone regeneration by stimulating the osteogenic differentiation of endogenous stem cells. Int J Biol Macromol. 2023;243:125330. doi:10.1016/j.ijbiomac.2023.125330\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003e Hu J, Miszuk JM, Stein KM, Sun H. Nanoclay promotes mouse cranial bone regeneration mainly through modulating drug binding and sustained release. Appl Mater Today. 2020;21:100860. doi:10.1016/j.apmt.2020.100860\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003e Huang K, Ou Q, Xie Y, Chen X, Fang Y, Huang C, et al. Halloysite nanotube based scaffold for enhanced bone regeneration. ACS Biomater Sci Eng. 2019;5(8):4037\u0026ndash;4047. doi:10.1021/acsbiomaterials.9b00277\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003e Li H, Mao B, Zhong J, Li X, Sang H. Localized delivery of metformin via 3D printed GelMA-Nanoclay hydrogel scaffold for enhanced treatment of diabetic bone defects. J Orthop Translat. 2024;47:249\u0026ndash;260. doi:10.1016/j.jot.2024.06.013\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003e Basaran SH, Bayrak A, Tanrıverdi G, Tanriverdi B, Avkan MC. Partial load-bearing rabbit ulnar segmental defects are regenerated with biocompatible grafts with or without bone marrow-derived mesenchymal stem cells. Ulus Travma Acil Cerrahi Derg. 2022;28(8):1066\u0026ndash;1072. doi:10.14744/tjtes.2021.64569\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003e Salehi S, Ghomi H, Hassanzadeh-Tabrizi SA, Koupaei N, Khodaei M. 3D printed polylactic acid/polyethylene glycol/bredigite nanocomposite scaffold enhances bone tissue regeneration via promoting osteogenesis and angiogenesis. Int J Biol Macromol. 2024;281(Pt 1):136160. doi:10.1016/j.ijbiomac.2024.136160\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003e Zhang W, Zhang Y, Zhang A, Ling C, Sheng R, Li X, et al. Enzymatically crosslinked silk-nanosilicate reinforced hydrogel with dual-lineage bioactivity for osteochondral tissue engineering. Mater Sci Eng C Mater Biol Appl. 2021;127:112215. doi:10.1016/j.msec.2021.112215\u003c/span\u003e\u003c/li\u003e\u003c/ol\u003e"}],"fulltextSource":"","fullText":"","funders":[],"hasAdminPriorityOnWorkflow":false,"hasManuscriptDocX":true,"hasOptedInToPreprint":true,"hasPassedJournalQc":"","hasAnyPriority":false,"hideJournal":false,"highlight":"","institution":"","isAcceptedByJournal":false,"isAuthorSuppliedPdf":false,"isDeskRejected":"","isHiddenFromSearch":false,"isInQc":false,"isInWorkflow":false,"isPdf":false,"isPdfUpToDate":true,"isWithdrawnOrRetracted":false,"journal":{"display":true,"email":"[email protected]","identity":"bmc-musculoskeletal-disorders","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":false,"externalIdentity":"bmsd","sideBox":"Learn more about [BMC Musculoskeletal Disorders](http://bmcmusculoskeletdisord.biomedcentral.com/)","snPcode":"","submissionUrl":"https://author-welcome.nature.com/12891","title":"BMC Musculoskeletal Disorders","twitterHandle":"BMC_series","acdcEnabled":true,"dfaEnabled":true,"editorialSystem":"stoa","reportingPortfolio":"BMC Series","inReviewEnabled":true,"inReviewRevisionsEnabled":true},"keywords":"Bone regeneration, Critical-size bone defect, Polylactic acid, Nanoclay, Biodegradable scaffold, NK-75, Lane–Sandhu score, Rabbit ulnar model, Bone tissue engineering, Nanocomposite","lastPublishedDoi":"10.21203/rs.3.rs-8882301/v1","lastPublishedDoiUrl":"https://doi.org/10.21203/rs.3.rs-8882301/v1","license":{"name":"CC BY 4.0","url":"https://creativecommons.org/licenses/by/4.0/"},"manuscriptAbstract":"\u003ch2\u003eBackground\u003c/h2\u003e \u003cp\u003eCritical-size bone defects remain a formidable challenge in orthopaedic surgery. Biodegradable polymer scaffolds represent a promising alternative to autologous bone grafting, but conventional polylactic acid (PLA) scaffolds exhibit limited osteoconductivity and generate acidic degradation products unfavorable to bone healing. Incorporation of nanoclay particles into PLA matrices may overcome these limitations. This study evaluated a novel PLA/nanoclay (NK-75) biodegradable composite scaffold for healing critical-size bone defects in a rabbit ulnar model.\u003c/p\u003e\u003ch2\u003eMethods\u003c/h2\u003e \u003cp\u003eNew Zealand White rabbits underwent bilateral mid-diaphyseal ulnar osteotomy creating 3 mm extraperiosteal critical-size defects. Defects were assigned to three groups: PLA\u0026thinsp;+\u0026thinsp;NK-75 composite scaffold (n\u0026thinsp;=\u0026thinsp;15 observations), PLA-only scaffold (n\u0026thinsp;=\u0026thinsp;4), or untreated empty defect control (n\u0026thinsp;=\u0026thinsp;11). Animals were sequentially sacrificed at 4, 8, 12, 16, and 20 weeks post-surgery. Radiological healing was assessed using the Lane-Sandhu scoring system (total score 0\u0026ndash;10, comprising bone formation, proximal union, distal union, and remodeling). Data were analyzed using Kruskal-Wallis test with Dunn's post-hoc comparisons, Mann-Whitney U test, linear mixed models, generalized estimating equations, ordinal logistic regression, and permutation tests. Effect sizes and post-hoc power were calculated.\u003c/p\u003e\u003ch2\u003eResults\u003c/h2\u003e \u003cp\u003eThe PLA\u0026thinsp;+\u0026thinsp;NK-75 group demonstrated significantly higher mean total Lane-Sandhu scores (7.20\u0026thinsp;\u0026plusmn;\u0026thinsp;1.93) compared to PLA (4.75\u0026thinsp;\u0026plusmn;\u0026thinsp;3.59) and control (4.00\u0026thinsp;\u0026plusmn;\u0026thinsp;2.32; Kruskal-Wallis p\u0026thinsp;=\u0026thinsp;0.0144). Post-hoc analysis confirmed PLA\u0026thinsp;+\u0026thinsp;NK-75 superiority over control (Dunn's p\u0026thinsp;=\u0026thinsp;0.0062; Mann-Whitney p\u0026thinsp;=\u0026thinsp;0.0044). Large effect sizes were observed (Hedges' g\u0026thinsp;=\u0026thinsp;1.47; Cliff's delta\u0026thinsp;=\u0026thinsp;0.66). The probability that a randomly selected PLA\u0026thinsp;+\u0026thinsp;NK-75 observation exceeds a control observation was 83.0%. Excellent healing (score\u0026thinsp;\u0026ge;\u0026thinsp;8) was achieved in 46.7% of PLA\u0026thinsp;+\u0026thinsp;NK-75 observations versus 0% in controls (Fisher's exact p\u0026thinsp;=\u0026thinsp;0.0025). PLA\u0026thinsp;+\u0026thinsp;NK-75 was the only group demonstrating remodeling activity. A significant ordered trend (CONTROL\u0026thinsp;\u0026lt;\u0026thinsp;PLA\u0026thinsp;\u0026lt;\u0026thinsp;PLA\u0026thinsp;+\u0026thinsp;NK-75) was confirmed by the Jonckheere-Terpstra test (p\u0026thinsp;=\u0026thinsp;0.0021). Results were consistent across all parametric, non-parametric, and permutation-based analyses. Achieved statistical power was 85.5%.\u003c/p\u003e\u003ch2\u003eConclusion\u003c/h2\u003e \u003cp\u003eThe PLA/NK-75 biodegradable composite scaffold significantly enhances radiological bone healing with large effect sizes and accelerated milestone achievement. These findings support nanoclay-enhanced biodegradable scaffolds as a promising strategy for orthopaedic bone regeneration.\u003c/p\u003e","manuscriptTitle":"Efficacy of a novel polylactic acid/nanoclay (NK-75) biodegradable composite scaffold in healing critical-size bone defects: an experimental study in a rabbit ulnar model","msid":"","msnumber":"","nonDraftVersions":[{"code":1,"date":"2026-03-05 15:20:04","doi":"10.21203/rs.3.rs-8882301/v1","editorialEvents":[{"type":"communityComments","content":0},{"type":"editorInvitedReview","content":"","date":"2026-05-16T02:50:59+00:00","index":"hide","fulltext":""},{"type":"reviewerAgreed","content":"319592282974431208468708626521828384174","date":"2026-05-14T14:02:08+00:00","index":"hide","fulltext":""},{"type":"reviewerAgreed","content":"275781032486921512232015580702514654567","date":"2026-05-12T12:25:09+00:00","index":"hide","fulltext":""},{"type":"reviewersInvited","content":"","date":"2026-02-25T09:40:09+00:00","index":"","fulltext":""},{"type":"editorAssigned","content":"","date":"2026-02-25T09:38:43+00:00","index":"","fulltext":""},{"type":"editorInvited","content":"","date":"2026-02-23T15:00:52+00:00","index":"","fulltext":""},{"type":"checksComplete","content":"","date":"2026-02-21T07:19:13+00:00","index":"","fulltext":""},{"type":"submitted","content":"BMC Musculoskeletal Disorders","date":"2026-02-21T06:36:55+00:00","index":"","fulltext":""}],"status":"published","journal":{"display":true,"email":"[email protected]","identity":"bmc-musculoskeletal-disorders","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":false,"externalIdentity":"bmsd","sideBox":"Learn more about [BMC Musculoskeletal Disorders](http://bmcmusculoskeletdisord.biomedcentral.com/)","snPcode":"","submissionUrl":"https://author-welcome.nature.com/12891","title":"BMC Musculoskeletal Disorders","twitterHandle":"BMC_series","acdcEnabled":true,"dfaEnabled":true,"editorialSystem":"stoa","reportingPortfolio":"BMC Series","inReviewEnabled":true,"inReviewRevisionsEnabled":true}}],"origin":"","ownerIdentity":"b7a039b1-fa9e-479b-8740-33d56f9ad993","owner":[],"postedDate":"March 5th, 2026","published":true,"recentEditorialEvents":[{"type":"editorInvitedReview","content":"","date":"2026-05-16T02:50:59+00:00","index":45,"fulltext":""},{"type":"reviewerAgreed","content":"319592282974431208468708626521828384174","date":"2026-05-14T14:02:08+00:00","index":43,"fulltext":""},{"type":"reviewerAgreed","content":"275781032486921512232015580702514654567","date":"2026-05-12T12:25:09+00:00","index":40,"fulltext":""}],"rejectedJournal":[],"revision":"","amendment":"","status":"under-review","subjectAreas":[],"tags":[],"updatedAt":"2026-03-05T15:20:04+00:00","versionOfRecord":[],"versionCreatedAt":"2026-03-05 15:20:04","video":"","vorDoi":"","vorDoiUrl":"","workflowStages":[]},"version":"v1","identity":"rs-8882301","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-8882301","identity":"rs-8882301","version":["v1"]},"buildId":"XKTyCvWXoU3ODBz1xrDgd","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}