Small-Diameter and Large-Depth Microfracture Improves Cartilage Repair in the Rabbit Osteochondral Defect Model

Small-Diameter and Large-Depth Microfracture Improves Cartilage Repair in the Rabbit Osteochondral Defect 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 Small-Diameter and Large-Depth Microfracture Improves Cartilage Repair in the Rabbit Osteochondral Defect Model Zhengbo Yin, Yifei Zhu, Zhian Chen, Hui Fang, Ni Yin, Youhua Cheng, and 3 more This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-5532606/v1 This work is licensed under a CC BY 4.0 License Status: Posted Version 1 posted You are reading this latest preprint version Abstract Previous studies have confirmed that small-diameter microfracture improves articular cartilage repair more effectively than do large-diameter microfracture and that drilling more deeply improves repair tissue quality. However, in microfracture (MF) surgery, the optimal diameters and depths of the holes drilled into the subchondral bone and their influence on cartilage healing are currently unknown. This study established a rabbit osteochondral defect model and treated cartilage lesions with modified microfracture (MMF) applications of different diameters and depths. Cartilage repair was detected through gross observation, histological analyse and immunohistochemical analyse. The results showed that MF with a diameter of 0.4 mm and a depth of 9 mm enhanced cartilage regeneration at 6 weeks after creating an osteochondral defect and resulted in virtually normal cartilage healing at 12 weeks. The repaired cartilage in the MMF group (diameter 0.4 mm and depth 9 mm) was more hyaline-like than those in the defect and other microfracture groups. In summary, this study confirmed that small-diameter and large-depth MMF could promote cartilage repair. Microfracture Osteochondral defect Cartilage repair Diameter Depth Figures Figure 1 Figure 2 Figure 3 Figure 4 Figure 5 Figure 6 Figure 7 Figure 8 Introduction Articular cartilage lesions are widespread, affecting approximately 1 million individuals in the United States [ 1 ] . Such defects can induce pain and lead to decreased mobility and eventually osteoarthritis, underlining a significant strain on the health care system [ 2 – 4 ] . Articular surface debridement with chondral shaving, abrasion chondroplasty, microfracture techniques, soft tissue arthroplasties such as periosteal and perichondrial grafts, and chondrocyte or osteochondral transplantation are among the current treatment options for articular cartilage injuries [ 5 – 9 ] . Microfracture is currently the most widely utilized technique to treat cartilage defects in clinical practice because of its low cost, low morbidity profile, and convenience of use [ 9 ] . Microfracture(MF) is an arthroscopic marrow stimulation technique that represents a key first-line treatment option for symptomatic small cartilage defects [ 10 ] and was developed by Dr Richard Steadman approximately 25 years ago [ 11 ] . The surgical procedure consists of removing the calcified layer [ 12 ] and creating small controlled holes through the subchondral bone to reach the cancellous bone [ 13 , 14 ] . Marrow clots fill the chondral defects and are believed to provide bone marrow mesenchymal stem cells (BMSCs), resulting in chondrogenic differentiation of mesenchymal stem cells in the cartilage repair tissue [ 15 , 16 ] . Gradually, fibrocartilaginous repair tissue fills the entire defect [ 1 , 17 , 18 ] . The mechanism of cartilage repair using bone marrow stimulation techniques has been well demonstrated [ 19 – 25 ] . Many studies have investigated the effects of the number of perforations in the defect region, the variety of instruments used for subchondral perforation, the depth of perforation, and the width of the subchondral perforator instrument on MSC mobilization for cartilage healing [ 26 – 29 ] . As demonstrated in previous studies, diameter and depth have substantial impacts on bone marrow stimulation [ 30 , 31 ] . Marchand et al. [ 32 ] observed no differences when full-thickness chondral lesions in the rabbit trochlea were repaired with either 0.5- or 0.9-mm holes at 6 months postoperatively. In contrast, Eldracher et al. [ 29 ] conducted research on an animal model to evaluate the healing of chondral defects by means of macroscopic, histological, immunohistochemical, and microcomputed tomography examinations treated using a perforation diameter of either 1 or 1.8 mm. The results were unambiguously better for defects treated with holes with smaller diameters and resulted in repaired tissue with a greater proportion of type II collagen and an architecture that was more comparable to that of normal tissue. Orth et al. [ 33 ] used two custom-made awls with a diameter of either 1 or 1.2 mm, and the depth was stopped at 5 mm. The authors observed that utilizing tiny-diameter awls resulted in a considerable improvement in overall histology scores. In addition, smaller instruments greatly enhanced the regularity of the histological surface. However, in young rabbits, the size of the aperture of the subchondral bone plate has a proportionate effect on the quantity of recruited mesenchymal stem cells, presumably accelerating cartilage regeneration [ 34 ] . Kok et al. [ 35 ] treated talar osteochondral lesions in a caprine model with microfracture holes 0.45 or 1.10 mm in diameter and 2 or 4 mm in depth, and after 4 months, no significant influence of perforation size on cartilage healing was observed. Nonetheless, Chen and colleagues conducted many investigations assessing the histological features of the newly generated tissue subsequent to chondral defect therapy using diverse repair methods and demonstrated varying levels of penetration into the subchondral bone [ 26 – 28 ] . Histological findings revealed that the best access to bone marrow was obtained when drilling into deeper areas, as this led to improved defect filling and the production of cartilage with a higher hyaline content [ 27 ] . In addition, Benthien et al. introduced the nanofracture technique using smaller-diameter and deeper subchondral bone needle perforations to produce cartilage with greater hyaline content [ 36 ] . Oddly enough, there are no precise guidelines regarding the depth or hole diameter for subchondral drilling. To further clarify the roles of microfracture hole diameter and depth in the repair of full-thickness osteochondral defects, we induced full-thickness cartilage defects at two distinct diameters and depths in a preclinical rabbit model. We specifically examined the hypothesis that osteochondral repair is improved when the subchondral bone is perforated with a small-diameter and large-depth device. To rule out other factors that could interfere with the repair process, we applied only one drill hole size per defect and the same number of drill holes for each treatment. We also included a group of defects with simple debridement of subchondral bone to investigate the effect of microfracture itself. Materials and Methods 2.1 Experimental animals The use of animals followed the Committee for Animal Use of the 920th Hospital of the PLA Joint Logistics Support Force (Ethics 2022-072 (Department)-01). Sixty healthy New Zealand White rabbits, aged 4 months and weighing approximately 3 ± 0.2 kg, with no sex limit, were purchased from Kunming Kejing Co., Ltd. The rabbits were randomly divided into 5 groups (n = 12 each): (1) osteochondral defect (Group D); (2) osteochondral defect + diameter 0.4 mm and depth 2 mm MF (Group A1); (3) osteochondral defect + diameter 0.4 mm and depth 9 mm MF (Group A2); (4) osteochondral defect + diameter 0.7 mm and depth 2 mm MF (Group B1); and (5) osteochondral defect + diameter 0.7 mm and depth 9 mm MF (Group B2) (Table 1 ). Before the study began, the animals were kept under standard laboratory conditions. 2.2 Microfracture surgery Sixty rabbits were randomly selected for the right or left knee joints using a computer and anaesthetized with pentobarbital sodium (3%, 1 mL/kg; Shandong Huamu Pharmaceutical Co., Ltd.). After successful anaesthesia, an osteochondral defect (diameter, 5 mm; depth, 2 mm) was created in the distal femur (trochlear groove) of 1 limb in all 60 rabbits using a trephine drill as previously described [ 37 ] . Microfracture was performed immediately after the osteochondral defect was created using 0.7 and 0.4 mm drill bits at depths of 2 and 9 mm respectively, with a density of 5 bur holes per 5 mm of osteochondral defect, as previously described [ 30 , 38 ] . An osteochondral defect rather than a chondral defect was created, as previous animal studies have shown that when only cartilage is damaged in rabbits, it is possible to for the damage to heal without treatment [ 39 , 40 ] . The 2-mm depth of the defect resulted in partial removal of the subchondral cortical bone, and it did not reach the cancellous bone. All procedures were carefully performed, and the tools were cooled with water to prevent heat damage to the cartilage and bone. Care was taken to preserve the miniclots formed on the microfracture holes. Drilling was chosen instead of using an awl because recent literature has shown its superiority to microfracture using an awl. Furthermore, drilling is more reproducible with regard to depth and location, particularly in small-animal models [ 41 ] . The rabbits were randomly distributed into 5 groups (n = 12 per group) as follows: Groups D, A1, A2, B1, and B2 (Table 1 ). The rabbits were fed standard feed and water ad libitum. All rabbits were allowed to walk freely in their cages with no movement restrictions. Veterinary staff from the Animal Research Laboratory of a tertiary hospital provided standard postoperative care for the rabbits, including infection prevention, pain reduction and wound observation. All rabbits were sacrificed at either 6 or 12 weeks after surgery for analysis. Table 1 Description of Groups Groups n Procedure D group 12 osteochondral defect only(5 mm in diameter and 2 mm in depth) A1 group 12 osteochondral defect + MF (0.4 mm in diameter and 2 mm in depth) A2 group 12 osteochondral defect + MF (0.4 mm in diameter and 9 mm in depth) B1 group 12 osteochondral defect + MF (0.7 mm in diameter and 2 mm in depth) B2 group 12 osteochondral defect + MF (0.7 mm in diameter and 9 mm in depth) 2.3 Macroscopic Analysis The distal femur was immediately dissected after euthanasia as previously described [ 56 ] . Two orthopaedists and two histologists performed gross observations in a blinded manner, and the results were evaluated using the International Cartilage Repair Society (ICRS) scoring system [ 42 ] . The ICRS scores were averaged from the separate evaluations. All excised distal femurs were photographed with a digital camera. 2.4 Micro-CT To avoid desiccation during scanning, parafilm was placed over the distal femurs after fixation. A 3-dimensional SkyScan 1276 Micro-CT (Bruker micro-CT, Kontech, Belgium) was used to scan the femurs with the following parameters: voltage, 100 kV; current, 200 µA; AI + Cu filter; voxel size, 20 µm; and rotation step, 0.4°. The images were reconstructed using NRecon software (Bruker micro-CT, Kontech, Belgium) with the following settings: the annular artefact was corrected to 5, and smoothing was applied to 3. In addition, 200 slices (4 mm) in the middle of each defect area were analysed, and the same threshold (cross-sectional view) was utilized to evaluate subchondral bone healing [ 56 ] . 2.5 Histological Analysis After decalcification, the tissues were subjected to a series of gradations in alcohol to remove moisture, followed by xylene clearing, processing, and paraffin embedding. Each paraffin block was attached to the cutting device, the slice thickness was set at 4 µm, paraffin slices were made, and water was used to dewax the slices. The sections were subjected to haematoxylin and eosin (G1120, Solarbio, Shanghai, China), Safranin O (G1371, Solarbio, Shanghai, China) and Alcian blue (G1560, Solarbio, Shanghai, China) staining. A fluorescence microscope (Nikon Eclipse E100, Nikon, Japan) was used for microscopic examination, and panoramic scanning was performed using an imaging system (Nikon DS-U3, Nikon, Japan) for image acquisition and analysis. Cartilage repair was assessed through the modified O’Driscoll and ICRS grading systems [ 43 ] based on Alcian blue and Safranin O staining. 2.6 Immunohistochemical Analysis Immunohistochemistry was carried out according to previously reported methods [ 56 ] . The sections were deparaffinized using xylene and gradient ethanol, rehydrated with deionized water, and then incubated for 30 minutes at room temperature with 2% hyaluronidase (H3506-5G; Sigma) in phosphate-buffered saline (pH 7.4) for antigen retrieval. Next, the sections were incubated with anti-type 1 collagen (1:500, 14695-1-AP; Proteintech Group, Wuhan, China) and anti-type 2 collagen (1:500, 28459-1-AP; Proteintech Group, Wuhan, China) overnight, and PBS was used to wash away the residue antibody. Afterwards, the sections were incubated with a goat anti-rabbit secondary antibody (K5007, Dako, Shanghai, China) conjugated with HRP for 50 minutes. Then, diaminobenzidine (DA1016, Solarbio, Shanghai, China) was used to develop the colour, and nuclear counterstaining was performed with haematoxylin. After being dehydrated, the slides were sealed with neutral gum. Finally, a fluorescence microscope (Nikon Eclipse E100, Nikon, Japan) was used to observe the sections, and an imaging system (Nikon DS-U3, Nikon, Japan) was used to perform panoramic scanning and collect the images. The positive ratio of type I collagen to type II collagen was calculated using ImageJ software (version 1.8.0.345) after image acquisition, and the different diameter and depth MMF groups were analysed for significant differences from the blank control group. 2.7 Statistical Analysis Continuous variables are presented as the mean ± standard deviation (SD) and were analysed by IBM SPSS Statistics 26.0 statistical software. The positive area ratios of type I collagen and type II collagen were calculated using ImageJ (version 1.8.0.345). ANOVA was used for statistical analysis, followed by Tukey’s post hoc test for two-group comparisons. The data were analysed using GraphPad Prism (version 10.0). A value of P < 0.05 was considered statistically significant. Results 3.1 Micro-CT, gross observation and ICRS score assessment of subchondral bone healing 6 weeks after surgery Six weeks postoperatively, gross observation revealed that the cartilage in the defect area in Group A2 had essentially healed, and the colour was nearly the same as that of the surrounding host cartilage. The defect areas were poorly healed in Groups D and A1, with small amounts of regenerated cartilage formed in the defect areas. Groups B1 and B2 exhibited incomplete cartilage healing in the defect areas, though the colour of the regenerated cartilage was essentially the same as that of the surrounding cartilage (Fig. 1 c). We performed micro-CT of the damaged bones, and the reconstructed images showed that the subchondral bone defects in Group A2 had essentially healed. However, the subchondral bone damage had not healed in Groups A1, B1, B2 and D, and the healing of the microfracture holes was incomplete (Fig. 1 a). A cross-sectional view of the middle area of the defect further showed that subchondral bone healing was greatest in Group A2. Small amounts of bone trabeculae were observed in the subchondral bone in Groups B1 and B2, and Groups D and A1 had more obvious subchondral bone defect areas and no significant bone trabecular regeneration (Fig. 1 b). Group A2 had the highest ICRS score, as determined by macroscopic evaluation, and the score was significantly greater than that in Group D (Fig. 1 d). 3.2 H&E staining and Alcian blue staining were used to evaluate the effects of cartilage repair We performed histological analyses on the defect areas of the distal femur cartilage. H&E staining revealed very light pink-stained cartilage layers in all groups. In Groups A1, A2, B1 and B2, the microfracture hole areas were thickened, and regenerated cartilage extended to the subchondral bone. The surface of the regenerated cartilage of Group B1 was not stained uniformly. Full cartilaginous covering of the defect (weak staining), which was morphologically very thick in the groove area, was observed in Group A1. Compared with those in Group A1, the cartilage in both Groups A2 and B2 appeared more like hyaline cartilage, demonstrating well-regenerated subchondral bone with pink bone matrix and less thickened cartilage. The surface of the regenerated cartilage of Group B1 was not stained uniformly. However, in Group D, less cartilage had regenerated in the defect area (Fig. 2 a). Alcian blue staining showed that the regenerated cartilage areas in Groups A1 and B1 were similar. Both groups displayed hypertrophy and overgrowth, with focal areas of subchondral bone formation, fibrillation, and cysts. In both Groups A2 and B2, the cartilage exhibited normal structural characteristics with relatively thin blue matrix. However, in Group D, no blue cartilage matrix was observed in the defect area, while the growth plate showed robust blue staining, indicating the absence of regenerated cartilage (Fig. 2 b) 3.3 Histology Scores Demonstrated Optimal Cartilage Repair in the A2 Group We further performed Safranin O staining for glycosaminoglycans (GAGs), an important structural component of the cartilage matrix. The repaired cartilage in Groups B1 and B2 demonstrated weak Safranin O positivity without full regeneration of the subchondral bone and still showed orange‒red staining. In contrast, Groups A2 and A1 showed robust orange‒red staining at the cartilage surface. In Group D, the defect demonstrated no signs of healing, with only subchondral bone and fibrotic tissues observed at the defect site. The growth plate cartilage was stained orange‒red (Fig. 3 a).. On O’Driscoll scoring, Group A2 demonstrated a significantly greater score than did Groups A1, B1, B2 and D. Group A1, A2, B1, B2 and D were analysed by ANOVA, and the differences were statistically significant (p < 0.001) (Fig. 3 b). 3.4 Immunohistochemical Assessments of type 1 collagen (Col-1) and type 2 collagen (Col-2) Six weeks after surgery, we examined the expression level of Col-1, which is related to fibrocartilage. The brown indicates collagen I, and at 20× magnification, a large amount of collagen I staining was observed on the surface of regenerated cartilage in all MMF groups; high expression was observed in Groups A1, A2, B1 and B2; subchondral bone was not completely healed in all groups; and a small amount of collagen I-positive cartilage was observed in Group D. At 100× magnification, fibrosis on the surface of regenerated cartilage-central subchondral bone was observed in all MMF groups, and the fibrosis in Group A2 was less than those in the other groups. The ratio of collagen I-positive cells in Group B1 was the highest, and paired t tests between Group D and Groups A1, A2, B1, and B2 revealed that the values conformed to a normal distribution, and the differences were statistically significant (P < 0.001) (Fig. 4 a and Fig. 4 b). We performed immunohistochemistry to measure Col-2. Brown indicates Col-2, and at 20× magnification, a small amount of brown collagen II staining was observed on the surface of regenerated cartilage in the MMF groups, and the regenerated cartilage in the cartilage defect area was not completely filled. There was more collagen II-positive cartilage in Group A2 than in the other groups. Subchondral bone staining was intense in Group A2, and it was not completely healed in Group B2. A very small amount of collagen II-positive cartilage was observed in Group D. The regenerated cartilage surfaces in Groups A1 and B2 appeared to be stripped and poorly junctioned, and negative collagen II staining of the cartilage surface and a large number of poor junctions on the cartilage surface were observed in Group D. The positive ratio of collagen II was highest in Group A2, and the paired t test between Group D and Groups A1, A2, B1, and B2 showed that the values conformed to a normal distribution, and the differences were statistically significant (P < 0.001) (Fig. 4 c and Fig. 4 d). 3.5 Gross observation and ICRS score assessment of subchondral bone healing 12 weeks after surgery At 12 weeks postoperative, gross observation revealed that the cartilage in the defect areas in the MMF groups had essentially healed, and the colour of Group A2 was basically the same as that of the surrounding host cartilage. The difference in the colour of the regenerated cartilage between Groups A1 and B2 and the surrounding cartilage was greater than that of Group B1, the central point of regenerated cartilage in the defect area of Group B2 had a dotted depression, and the colour of regenerated cartilage in the defect area of Group D had a greater difference in the colour of the regenerated cartilage compared with that of the surrounding host cartilage (Fig. 5 a). Group A2 had the highest ICRS score, as determined by macroscopic evaluation, and the score was significantly greater than that in Group D. Analysis of variance revealed that the differences among Groups A1, A2, B1, B2 and D were statistically significant (P < 0.001). (Fig. 5 b). 3.6 H&E staining and Safranin O staining to evaluate cartilage repair 12 weeks after surgery Twelve weeks after surgery, compared with those in Groups A1, B1 and B2, the regenerated cartilage in Group A2 was thicker, the interface between the new cartilage and host cartilage was more fully integrated, the cartilage and subchondral bone were evenly distributed, and the cartilage was hyaline cartilage-like at 12 weeks after the operation, while one side of the subchondral bone was not completely healed in Group B1, the central subchondral bone was not healed in Group B2, and the central regenerated cartilage was missing in Group D. (Fig. 6 a). In Alcian blue staining, the nucleus is stained red, and the cytoplasm is stained light red. The cartilage layer was not connected continuously, the cartilage surface was obviously fibrotic, a few adipocytes were observed in the cartilage layer, and the defect area also showed light blue cartilage. In Groups A1 and B2, deep blue cartilage was observed from the cartilage layer to the subchondral bone, a small amount of adipocytes was noted in the cartilage layer, and a small amount of fibrosis was observed on the surface of the cartilage. In Group A2, the morphology of regenerated cartilage was normal, and dark blue cartilage was noted from the centre of the cartilage layer to the subchondral bone. In Group D, there was a large amount of fibrosis in the central part of the cartilage defect, a very small amount of dark blue cartilage and a large number of adipocytes in the cartilage layer (Fig. 6 b). 3.7 Safranin O staining and O’Driscoll scores for evaluating cartilage repair 12 weeks after surgery After 12 weeks, we carried out Safranin O staining. At 20× magnification, the middle part of the regenerated cartilage to the subchondral bone in the MF groups were Safranin O positive, and the surface of the cartilage was colourless. Compared with those in Groups A1 and B1, the repaired cartilage showed strong orange‒red staining in Group A2, strong orange‒red staining in Group B2 and no orange‒red staining in the centre of regenerated cartilage in Group D(Fig. 7 a). The histological O'Driscoll scores of all the samples were evaluated 12 weeks after the operation, and the score in Group A2 was the highest. Groups A1, A2, B1, B2 and D were analysed by ANOVA, and the differences were statistically significant (p < 0.001) (Fig. 7 b). 3.8 Immunohistochemical assessment of Col-1 and Col-2 12 weeks postoperatively Large amounts of collagen I staining were observed on the surface of regenerated cartilage in the MF groups, while high expression was detected in Groups A1 and B1. The staining ratio in Group A2 at 12 weeks was significantly lower than that at 6 weeks. Subchondral bone had not completely healed in any of the groups, and a very small amount of collagen I-positive cartilage was found in Group D. Fibrosis on the surface of regenerated cartilage-central subchondral bone was observed in all the MF groups, though there was less fibrosis in Group A2 than in the other groups (Fig. 8 a). The ratio of collagen I-positive cells in Group D was the highest, and paired t tests between Group D and Groups A1, A2, and B1 revealed that the values conformed to a normal distribution, and the differences were statistically significant (P < 0.001). (Fig. 8 a and Fig. 8 b). In type II collagen staining, brown indicates collagen II. A small amount of brown collagen II staining was observed on the surface of the regenerated cartilage in each of the MMF groups, and the regenerated cartilage in the cartilage defect areas was almost completely filled. Compared with Groups A1, B1 and B2, there was more collagen II-positive cartilage in Group A2, but subchondral bone was strongly stained and did not heal completely in Groups A2 and B2, and there was very little collagen II-positive cartilage in Group D. Among the MMF groups, collagen II staining in the cartilage region was evident in Group A2, which had tightly connected cartilage surfaces and a small number of cracks on the cartilage surfaces; the regenerated cartilage surfaces were not tightly connected in Groups A1 and B2; and negative collagen II staining of the cartilage surfaces and a large number of cracks on the cartilage surfaces were observed in Group D (Fig. 8 c). The positive ratio of collagen II in Group A2 was the highest, and the paired t test between Group D and Groups A1 and A2 revealed that the values conformed to a normal distribution, and the differences were statistically significant (P < 0.001) (Fig. 8 c and Fig. 8 d). Discussion The primary finding of the present study was that 0.4 mm-diameter and 9 mm-deep microfracture drill holes significantly improved articular cartilage repair compared to drill holes of larger diameter and smaller depth. Second, compared with control defects that were debrided alone, microfracture treatment involving the use of either drill holes of different diameters or depths led to better histological cartilage repair. The application of microfracture as a treatment option for cartilage abnormalities has been thoroughly examined in the literature, and various perforation techniques have been investigated [ 26 – 29 ] . However, the effects of different perforation diameters and depths on cartilage healing have not been well studied. In this experimental study, microfractures (subchondral perforations) from osteochondral defects of different diameters and depths were compared macroscopically, histologically, and immunohistochemically. The main findings of this study were that microfractures with different diameters and depths demonstrated different healing characteristics. The results showed that a diameter of 0.4 mm and a depth of 9 mm could significantly improve the repair of rabbit knee cartilage. Moreover, the content of hyaline cartilage in the repaired tissue was greater than those in the other groups. As a result, considerable effort has been made to develop and modify microfracture. Although MF has become the main option for therapy for cartilage lesions owing to its technical simplicity, minimal invasiveness, limited availability, low cost, and autogenous nature, which enhance cartilage repair by releasing BMSCs through small drill holes in cartilage defects [ 38 , 44 ] , the long-term efficacy of MF was weak in many follow-up examinations due to the production of more fibrocartilage after the procedure [ 44 – 46 ] . In addition, several alternative treatment modalities have emerged to address focal chondral defects that could promote hyaline cartilage repair, including particulate cartilage allografts [ 47 , 48 ] , osteochondral allografts or autografts [ 49 ] , and hyaline-like cartilage such as autologous chondrocyte implantation [ 50 ] . Microfracture techniques to alter bone marrow stimulation for the purpose of regenerating cartilage similar to normal hyaline cartilage, and different cartilage repair effects have been shown by varying the diameter, depth, number of drilled holes, spacing of holes, location, and other characteristics of microfractures alone [ 27 , 29 , 33 , 51 – 53 ] . However, several of these modified microfracture approaches have yet to be utilized in clinical. Interestingly, the effects of perforations of different diameters and depths on osteochondral healing have received little research attention. Few studies have examined the impacts of microfracture drill hole diameter and depth on osteochondral repair. In a small animal model, no differences were reported when full-thickness cartilage defects in the trochlea were subjected to drilling using either 0.5-mm (proximal trochlea) or 0.9-mm (distal trochlea) holes at 6 months postoperatively [ 32 ] . The application of 1.0-mm subchondral drill holes significantly improved histological articular cartilage repair compared to 1.8-mm holes, as indicated by their better average total score. Moreover, matrix staining for Safranin O was significantly increased, suggesting that the repair tissue in the 1.0-mm drill holes contained more proteoglycans. In addition, the number of cells resembling articular chondrocytes within the repair of 1.0-mm drill holes was also greater, indicating that the structure of the repaired tissue more closely resembled that of cartilage. In good agreement with these findings, the immunoreactivity to type II collagen, an essential component of the normal cartilage extracellular matrix, was considerably greater in the tissue repaired with 1.0-mm drill holes. Conversely, the immunoreactivity to type I collagen (an indication of fibrocartilaginous repair) was considerably lower in the tissue repaired with 1.0-mm drill holes than in that repaired with 1.8-mm drill holes [ 29 ] . Min and colleagues [ 34 ] reported that larger holes (diameter of 1.5 mm) increase the access of BMSCs and growth factors to articular cartilage lesions in rabbit knee joints compared with smaller holes (0.8 mm). To date, only one study has investigated the influence of microfracture hole parameters on articular cartilage repair in vivo: Kok et al. [ 35 ] treated talar osteochondral lesions in a caprine model using microfracture holes 0.45 or 1.10 mm in diameter and 2 or 4 mm in depth. No significant effect of perforation size on cartilage repair was noted after 4 months. However, the applicability of the findings to the present research is limited due to significant differences between the ankle and knee joints [ 54 ] . Another investigation demonstrated significantly greater quantities of DNA (up to 3.9-fold), proteoglycans (up to 4.2-fold), and type II collagen (up to 4.0-fold) in cartilaginous repair tissue following small-diameter (1.0 mm) subchondral drilling than after large-diameter (1.8 mm) drilling [ 51 ] . This is consistent with our experimental results. Compared with those in the other groups, the amount of type I collagen in the 0.4 mm diameter sample was much lower, although the amount of type II collagen was significantly greater, demonstrating that an MF with a diameter of 0.4 mm repaired cartilage injuries to more closely resemble normal articular cartilage. Moreover, we discovered that the type I collagen contents were lowest in the defect group at 6 weeks after surgery and highest at 12 weeks after surgery, primarily due to the small amount of cartilage regeneration observed in the defect group at 6 weeks after surgery and the gradual regeneration of cartilage over time after 6 weeks. At 12 weeks, we observed a moderate amount of cartilage regeneration by gross observation and histology. Immunohistochemistry revealed that the regenerated cartilage in the defect group was predominantly type I collagen, which is consistent with the findings of osteochondral defect models established in previous studies [ 38 ] . The repair of articular cartilage injury by MF mainly depends on the recruitment of BMSCs [ 39 ] . Therefore, providing access to the bone marrow stroma is the first step in BMSC recruitment. Zedde et al. [ 30 ] conducted microfracture and nanomicrofracture in the sheep knee joint and discovered that nanomicrofracture promoted cartilage regeneration. Additionally, it has been observed that deeper (6 mm) subchondral drilling results in more hyaline cartilage repair than shallower (2 mm) drilling. This is evidenced by increased levels of glycosaminoglycan, type II collagen content, and cartilage defect filling, as well as a decrease in type I collagen [ 27 ] , which was more consistent with MF cartilage repair at a depth of 9 mm than at a depth of 2 mm in this study. Our findings suggest that microfracture with a diameter of 0.4 mm and a depth of 9 mm can considerably increase rabbit knee cartilage healing, possibly because the hole diameter and depth activate more BMSCs [ 51 ] . Reconstruction of the subchondral bone is essential for osteochondral repair [ 55 ] . Compared to 1.8-mm drilling, 1.0-mm drilling had normal bone density of the subchondral bone plate and subarticular cancellous bone, which was very comparable to that of the adjacent subchondral bone, and 1.0-mm drilling contributed to significantly better healing of the subchondral bone plate and a substantial decrease in subchondral bone plate thickening [ 33 ] . In our study, a drilling diameter of 0.4 mm caused less damage to the subchondral bone and led to better subchondral bone reconstruction and higher quality cartilage. In this study, we confirmed that different diameters and depths of MF had positive effects on cartilage repair and that MF with a diameter of 0.4 mm and a depth of 9 mm promoted the expression of type 2 collagen and the regeneration of hyaline cartilage. However, these parameters were chosen by considering previous studies to allow for legitimate comparisons [ 27 , 29 , 34 , 36 , 56 ] , which resulted in considerable differences between groups. Our research findings will motivate clinicians to explore viable therapeutic approaches for different diameters and depths of MF to enhance cartilage regeneration, addressing current clinical bottlenecks such as fibrocartilage formation and poor long-term outcomes after MF. We recognize that the current study is not without limitations. First, the 12-week follow-up period may be too short to provide enough clinical relevance. Second, the osteochondral defect area in the rabbit knees was created acutely rather than chronically. This makes the cartilage defect model created in animals less similar to articular cartilage damage in humans. Third, we only observed the condition of the subchondral bone by micro-CT at 6 weeks after surgery. Fourth, we did not measure the amount of BMSCs released after microfracture surgery. In addition, while the results from a rabbit osteochondral defect model are intriguing, further research in large animal models and eventually in humans should be conducted before clinical application. Finally, we did not perform a biomechanical evaluation of the regenerated cartilage tissue, but this may constitute future research. Conclusion MMF treatment significantly enhances cartilage repair, with the parameters of 0.4 mm in diameter and 9 mm in depth exhibiting the highest therapeutic efficacy. Declarations Acknowledgments This study is financed by Yunnan Science and Technology Department Key Projects(202401AY070001-045、202501AS070164) and Yunnan Orthopedics and Sports Rehabilitation Clinical Medicine Research Center (202102AA310068). Authors ’ Contributions H. T. and T.Z. conceived the study. Z.C. and Z.Y. performed all animal models. Y.X. performed CT analysis. N.Y. ,H.F. and Y.C. performed all histological tests. Z.Y. and Y.Z. wrote the paper. All authors have read and approved final version. Funding Declaration The authors disclose receipt of the following financial or material support for the research, authorship, and/or publication of this article: this work was supported by Yunnan Science and Technology Department Key Projects(202401AY070001-045、202501AS070164) and Yunnan Orthopedics and Sports Rehabilitation Clinical Medicine Research Center (202102AA310068). Data Availability All datasets presented in this study are included in the article. Conflicts of Interest The authors declare that they have no competing interests. References Mithoefer K, McAdams T, Williams R J, Kreuz, P C, Mandelbaum, B R. Clinical efficacy of the microfracture technique for articular cartilage repair in the knee: an evidence-based systematic analysis[J]. Am J Sports Med, 2009,37(10):2053-2063. Chen H, Chevrier A, Hoemann C D, Sun J, Lascau-Coman V, Buschmann M D. 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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-5532606","acceptedTermsAndConditions":true,"allowDirectSubmit":true,"archivedVersions":[],"articleType":"Research Article","associatedPublications":[],"authors":[{"id":444541246,"identity":"73859ad4-3979-4d98-b120-32069b1cc046","order_by":0,"name":"Zhengbo Yin","email":"","orcid":"","institution":"Yuxi Children's Hospital","correspondingAuthor":false,"prefix":"","firstName":"Zhengbo","middleName":"","lastName":"Yin","suffix":""},{"id":444541247,"identity":"7da2484b-5abb-4fd0-9c5c-3bcb0ac7e0a9","order_by":1,"name":"Yifei Zhu","email":"","orcid":"","institution":"Chenggong Hospital Affiliated KunmingYan’an Hospital","correspondingAuthor":false,"prefix":"","firstName":"Yifei","middleName":"","lastName":"Zhu","suffix":""},{"id":444541248,"identity":"bddda2de-5b95-49a7-ac73-507420e87c78","order_by":2,"name":"Zhian Chen","email":"","orcid":"","institution":"Kunming Medical University","correspondingAuthor":false,"prefix":"","firstName":"Zhian","middleName":"","lastName":"Chen","suffix":""},{"id":444541249,"identity":"45cb6526-97cc-4843-bc06-c30bb8df1e0b","order_by":3,"name":"Hui Fang","email":"","orcid":"","institution":"Yuxi Children's Hospital","correspondingAuthor":false,"prefix":"","firstName":"Hui","middleName":"","lastName":"Fang","suffix":""},{"id":444541250,"identity":"d652d296-516e-4c62-9cae-861f5ef08dc0","order_by":4,"name":"Ni Yin","email":"","orcid":"","institution":"The First Affiliated Hospital of Kunming Medical University","correspondingAuthor":false,"prefix":"","firstName":"Ni","middleName":"","lastName":"Yin","suffix":""},{"id":444541251,"identity":"378c0c1d-56cc-4625-a4f3-f723b1639ae3","order_by":5,"name":"Youhua Cheng","email":"","orcid":"","institution":"People’s Liberation Army Joint Logistic Support Force 920th Hospital","correspondingAuthor":false,"prefix":"","firstName":"Youhua","middleName":"","lastName":"Cheng","suffix":""},{"id":444541252,"identity":"2909ee05-3752-41b9-9335-1e58817a76f7","order_by":6,"name":"Yongqing Xu","email":"","orcid":"","institution":"People’s Liberation Army Joint Logistic Support Force 920th Hospital","correspondingAuthor":false,"prefix":"","firstName":"Yongqing","middleName":"","lastName":"Xu","suffix":""},{"id":444541253,"identity":"3e59a968-b192-468e-b50f-68c5b34ec85f","order_by":7,"name":"Tianhua Zhou","email":"","orcid":"","institution":"People’s Liberation Army Joint Logistic Support Force 920th Hospital","correspondingAuthor":false,"prefix":"","firstName":"Tianhua","middleName":"","lastName":"Zhou","suffix":""},{"id":444541254,"identity":"dc30f5d0-83da-4c0d-8296-22236221194a","order_by":8,"name":"Hongbo Tan","email":"data:image/png;base64,iVBORw0KGgoAAAANSUhEUgAAAZAAAAAyAQMAAABI0h/eAAAABlBMVEX///8AAABVwtN+AAAACXBIWXMAAA7EAAAOxAGVKw4bAAAAq0lEQVRIiWNgGAWjYDCCA0DM2GDDw8/fQJqWNBnJGQdI03LYxqAhgUgdfMebn0n+3HGex4DhAOOHjzlEaJE8c8xMQvLMbR5z5gZmyZnbiNBicCOHTcKw7TaPZcMBNmZeorUktp3jMTiQQIqWg20HSNAC9IuxZeOZZB7JGQebifMLMMQe3vy5w86en7/54IePxGgBAhYJCM3YQJx6IGD+QLTSUTAKRsEoGJkAANXuOVUDDcNMAAAAAElFTkSuQmCC","orcid":"","institution":"People’s Liberation Army Joint Logistic Support Force 920th Hospital","correspondingAuthor":true,"prefix":"","firstName":"Hongbo","middleName":"","lastName":"Tan","suffix":""}],"badges":[],"createdAt":"2024-11-27 06:38:38","currentVersionCode":1,"declarations":"","doi":"10.21203/rs.3.rs-5532606/v1","doiUrl":"https://doi.org/10.21203/rs.3.rs-5532606/v1","draftVersion":[],"editorialEvents":[],"editorialNote":"","failedWorkflow":false,"files":[{"id":80997305,"identity":"46b9d36d-6beb-4b15-8589-ad8a01da3be9","added_by":"auto","created_at":"2025-04-21 05:35:03","extension":"png","order_by":1,"title":"Figure 1","display":"","copyAsset":false,"role":"figure","size":754963,"visible":true,"origin":"","legend":"\u003cp\u003eMicro-CT and gross observation 6 weeks after surgery.(a)Micro-CT 3D images.(b)Cross-sectional view of the center of the osteochondral defect. (c,d)Gross image of osteochondral defect healing and the ICRS score. Asterisks indicate statistical significance (*p\u0026lt;0.05; **p\u0026lt;0.01; ***p\u0026lt;0.001).\u003c/p\u003e","description":"","filename":"Fig1.png","url":"https://assets-eu.researchsquare.com/files/rs-5532606/v1/18481b58f6f69967f3d278ab.png"},{"id":80998450,"identity":"dddfa194-17f4-46db-a433-9421ee0fdb7a","added_by":"auto","created_at":"2025-04-21 05:43:04","extension":"png","order_by":2,"title":"Figure 2","display":"","copyAsset":false,"role":"figure","size":1324857,"visible":true,"origin":"","legend":"\u003cp\u003eHematoxylin and eosin (H\u0026amp;E) and Alcian blue staining. (a) H\u0026amp;E Staining. (b) Alcian blue staining of osteochondral defect cartilage in the trochlear groove. Scale bar=500 µm and 100 µm at 20× magnification and 100× magnification, respectively.\u003c/p\u003e","description":"","filename":"Fig2.png","url":"https://assets-eu.researchsquare.com/files/rs-5532606/v1/a6064912f4205472861cc147.png"},{"id":80997307,"identity":"7cc98ff5-47f7-41e9-9261-21821f22c7cb","added_by":"auto","created_at":"2025-04-21 05:35:04","extension":"png","order_by":3,"title":"Figure 3","display":"","copyAsset":false,"role":"figure","size":589977,"visible":true,"origin":"","legend":"\u003cp\u003eSafranin O staining and histology score. (a) Safranin O staining of the trochlear groove cartilage. (b) O’Driscoll histology score.Analysis of variance (ANOVA), followed by the Tukey post hoc multiple comparison test(*p\u0026lt;0.05; **p\u0026lt;0.01; ***p\u0026lt;0.001) . Scale bar=500 µm and 100 µm at 20× magnification and 100× magnification, respectively.\u003c/p\u003e","description":"","filename":"Fig3.png","url":"https://assets-eu.researchsquare.com/files/rs-5532606/v1/a59ca5c2a1007c6e459a54b1.png"},{"id":80997346,"identity":"6cc84112-4e1d-4105-9ec2-35a665fca63b","added_by":"auto","created_at":"2025-04-21 05:35:06","extension":"png","order_by":4,"title":"Figure 4","display":"","copyAsset":false,"role":"figure","size":1757656,"visible":true,"origin":"","legend":"\u003cp\u003eImmunohistochemical analysis of collagen I and collagen II. (a) Staining for type I collagen. (b) Statistical chart showing the percentage of the type I collagen-positive area. (c) Staining for type II collagen. (d) The chart shows the percentage of the type II collagen-positive area. Scale bars=500 µm and 100 µm at 20× magnification and 100× magnification, respectively. Asterisks indicate statistical significance (*p\u0026lt;0.05; **p\u0026lt;0.01; ***p\u0026lt;0.001).\u003c/p\u003e","description":"","filename":"Fig4.png","url":"https://assets-eu.researchsquare.com/files/rs-5532606/v1/0a994015f44d71ac3f855118.png"},{"id":80997348,"identity":"8caffb10-93e2-4222-ac22-42884dcd4010","added_by":"auto","created_at":"2025-04-21 05:35:06","extension":"png","order_by":5,"title":"Figure 5","display":"","copyAsset":false,"role":"figure","size":543987,"visible":true,"origin":"","legend":"\u003cp\u003eGross images 12 weeks after surgery and ICRS scores.(a)Gross image of osteochondral defect healing.(b)the ICRS score. Asterisks indicate statistical significance (*p\u0026lt;0.05; **p\u0026lt;0.01; ***p\u0026lt;0.001).\u003c/p\u003e","description":"","filename":"Fig5.png","url":"https://assets-eu.researchsquare.com/files/rs-5532606/v1/d666558ab860bc55cd91f7bb.png"},{"id":80997325,"identity":"44e13fb9-3299-4533-9c9b-44acfabae0f4","added_by":"auto","created_at":"2025-04-21 05:35:05","extension":"png","order_by":6,"title":"Figure 6","display":"","copyAsset":false,"role":"figure","size":1372450,"visible":true,"origin":"","legend":"\u003cp\u003eHematoxylin and eosin (H\u0026amp;E) and Alcian blue staining. (a) H\u0026amp;E Staining. (b) Alcian blue staining of osteochondral defect cartilage in the trochlear groove. Scale bar=500 µm and 100 µm at 20×magnification and 100×magnification, respectively.\u003c/p\u003e","description":"","filename":"Fig6.png","url":"https://assets-eu.researchsquare.com/files/rs-5532606/v1/eac1eafeb9dc0814b146ee17.png"},{"id":80997319,"identity":"1d180d10-8155-46ae-bbc9-044262349818","added_by":"auto","created_at":"2025-04-21 05:35:04","extension":"png","order_by":7,"title":"Figure 7","display":"","copyAsset":false,"role":"figure","size":575463,"visible":true,"origin":"","legend":"\u003cp\u003eSafranin O staining and histology score. (a) Safranin O staining of the trochlear groove cartilage. (b) O’Driscoll histology score. Analysis of variance (ANOVA), followed by the Tukey post hoc multiple comparison test(*p\u0026lt;0.05; **p\u0026lt;0.01; ***p\u0026lt;0.001) . Scale bar=500 µm and 100 µm at 20×magnification and 100×magnification, respectively.\u003c/p\u003e","description":"","filename":"Fig7.png","url":"https://assets-eu.researchsquare.com/files/rs-5532606/v1/6f2584e2dfdcccc1dc21717b.png"},{"id":80998467,"identity":"733be342-17ca-4fa8-a02d-97cc25ea1da4","added_by":"auto","created_at":"2025-04-21 05:43:06","extension":"png","order_by":8,"title":"Figure 8","display":"","copyAsset":false,"role":"figure","size":1873204,"visible":true,"origin":"","legend":"\u003cp\u003eImmunohistochemical analysis of collagen I and collagen II. (a) Staining for type I collagen. (b) Statistical chart showing the percentage of the type I collagen-positive area. (c) Staining for type II collagen. (d) The chart shows the percentage of the type II collagen-positive area. Scale bars=500 µm and 100 µm at 20× magnification and 100× magnification, respectively. Asterisks indicate statistical significance (*p\u0026lt;0.05; **p\u0026lt;0.01; ***p\u0026lt;0.001).\u003c/p\u003e","description":"","filename":"Fig8.png","url":"https://assets-eu.researchsquare.com/files/rs-5532606/v1/b9c7857592be8a18ba39601b.png"},{"id":81481165,"identity":"b98ea2de-d401-4f4d-bd5c-4d3e4d1e4970","added_by":"auto","created_at":"2025-04-27 14:31:39","extension":"pdf","order_by":0,"title":"","display":"","copyAsset":false,"role":"manuscript-pdf","size":8708372,"visible":true,"origin":"","legend":"","description":"","filename":"manuscript.pdf","url":"https://assets-eu.researchsquare.com/files/rs-5532606/v1/3294635d-be0d-4e45-b4e1-4634a3c28bbe.pdf"}],"financialInterests":"No competing interests reported.","formattedTitle":"Small-Diameter and Large-Depth Microfracture Improves Cartilage Repair in the Rabbit Osteochondral Defect Model","fulltext":[{"header":"Introduction","content":"\u003cp\u003eArticular cartilage lesions are widespread, affecting approximately 1\u0026nbsp;million individuals in the United States\u003csup\u003e[\u003cspan citationid=\"CR1\" class=\"CitationRef\"\u003e1\u003c/span\u003e]\u003c/sup\u003e. Such defects can induce pain and lead to decreased mobility and eventually osteoarthritis, underlining a significant strain on the health care system\u003csup\u003e[\u003cspan additionalcitationids=\"CR3\" citationid=\"CR2\" class=\"CitationRef\"\u003e2\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR4\" class=\"CitationRef\"\u003e4\u003c/span\u003e]\u003c/sup\u003e. Articular surface debridement with chondral shaving, abrasion chondroplasty, microfracture techniques, soft tissue arthroplasties such as periosteal and perichondrial grafts, and chondrocyte or osteochondral transplantation are among the current treatment options for articular cartilage injuries\u003csup\u003e[\u003cspan additionalcitationids=\"CR6 CR7 CR8\" citationid=\"CR5\" class=\"CitationRef\"\u003e5\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR9\" class=\"CitationRef\"\u003e9\u003c/span\u003e]\u003c/sup\u003e. Microfracture is currently the most widely utilized technique to treat cartilage defects in clinical practice because of its low cost, low morbidity profile, and convenience of use\u003csup\u003e[\u003cspan citationid=\"CR9\" class=\"CitationRef\"\u003e9\u003c/span\u003e]\u003c/sup\u003e.\u003c/p\u003e \u003cp\u003eMicrofracture(MF) is an arthroscopic marrow stimulation technique that represents a key first-line treatment option for symptomatic small cartilage defects\u003csup\u003e[\u003cspan citationid=\"CR10\" class=\"CitationRef\"\u003e10\u003c/span\u003e]\u003c/sup\u003e and was developed by Dr Richard Steadman approximately 25 years ago\u003csup\u003e[\u003cspan citationid=\"CR11\" class=\"CitationRef\"\u003e11\u003c/span\u003e]\u003c/sup\u003e. The surgical procedure consists of removing the calcified layer\u003csup\u003e[\u003cspan citationid=\"CR12\" class=\"CitationRef\"\u003e12\u003c/span\u003e]\u003c/sup\u003e and creating small controlled holes through the subchondral bone to reach the cancellous bone\u003csup\u003e[\u003cspan citationid=\"CR13\" class=\"CitationRef\"\u003e13\u003c/span\u003e, \u003cspan citationid=\"CR14\" class=\"CitationRef\"\u003e14\u003c/span\u003e]\u003c/sup\u003e. Marrow clots fill the chondral defects and are believed to provide bone marrow mesenchymal stem cells (BMSCs), resulting in chondrogenic differentiation of mesenchymal stem cells in the cartilage repair tissue\u003csup\u003e[\u003cspan citationid=\"CR15\" class=\"CitationRef\"\u003e15\u003c/span\u003e, \u003cspan citationid=\"CR16\" class=\"CitationRef\"\u003e16\u003c/span\u003e]\u003c/sup\u003e. Gradually, fibrocartilaginous repair tissue fills the entire defect\u003csup\u003e[\u003cspan citationid=\"CR1\" class=\"CitationRef\"\u003e1\u003c/span\u003e, \u003cspan citationid=\"CR17\" class=\"CitationRef\"\u003e17\u003c/span\u003e, \u003cspan citationid=\"CR18\" class=\"CitationRef\"\u003e18\u003c/span\u003e]\u003c/sup\u003e. The mechanism of cartilage repair using bone marrow stimulation techniques has been well demonstrated\u003csup\u003e[\u003cspan additionalcitationids=\"CR20 CR21 CR22 CR23 CR24\" citationid=\"CR19\" class=\"CitationRef\"\u003e19\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR25\" class=\"CitationRef\"\u003e25\u003c/span\u003e]\u003c/sup\u003e. Many studies have investigated the effects of the number of perforations in the defect region, the variety of instruments used for subchondral perforation, the depth of perforation, and the width of the subchondral perforator instrument on MSC mobilization for cartilage healing\u003csup\u003e[\u003cspan additionalcitationids=\"CR27 CR28\" citationid=\"CR26\" class=\"CitationRef\"\u003e26\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR29\" class=\"CitationRef\"\u003e29\u003c/span\u003e]\u003c/sup\u003e. As demonstrated in previous studies, diameter and depth have substantial impacts on bone marrow stimulation\u003csup\u003e[\u003cspan citationid=\"CR30\" class=\"CitationRef\"\u003e30\u003c/span\u003e, \u003cspan citationid=\"CR31\" class=\"CitationRef\"\u003e31\u003c/span\u003e]\u003c/sup\u003e.\u003c/p\u003e \u003cp\u003eMarchand et al.\u003csup\u003e[\u003cspan citationid=\"CR32\" class=\"CitationRef\"\u003e32\u003c/span\u003e]\u003c/sup\u003e observed no differences when full-thickness chondral lesions in the rabbit trochlea were repaired with either 0.5- or 0.9-mm holes at 6 months postoperatively. In contrast, Eldracher et al.\u003csup\u003e[\u003cspan citationid=\"CR29\" class=\"CitationRef\"\u003e29\u003c/span\u003e]\u003c/sup\u003e conducted research on an animal model to evaluate the healing of chondral defects by means of macroscopic, histological, immunohistochemical, and microcomputed tomography examinations treated using a perforation diameter of either 1 or 1.8 mm. The results were unambiguously better for defects treated with holes with smaller diameters and resulted in repaired tissue with a greater proportion of type II collagen and an architecture that was more comparable to that of normal tissue. Orth et al.\u003csup\u003e[\u003cspan citationid=\"CR33\" class=\"CitationRef\"\u003e33\u003c/span\u003e]\u003c/sup\u003e used two custom-made awls with a diameter of either 1 or 1.2 mm, and the depth was stopped at 5 mm. The authors observed that utilizing tiny-diameter awls resulted in a considerable improvement in overall histology scores. In addition, smaller instruments greatly enhanced the regularity of the histological surface. However, in young rabbits, the size of the aperture of the subchondral bone plate has a proportionate effect on the quantity of recruited mesenchymal stem cells, presumably accelerating cartilage regeneration\u003csup\u003e[\u003cspan citationid=\"CR34\" class=\"CitationRef\"\u003e34\u003c/span\u003e]\u003c/sup\u003e. Kok et al.\u003csup\u003e[\u003cspan citationid=\"CR35\" class=\"CitationRef\"\u003e35\u003c/span\u003e]\u003c/sup\u003e treated talar osteochondral lesions in a caprine model with microfracture holes 0.45 or 1.10 mm in diameter and 2 or 4 mm in depth, and after 4 months, no significant influence of perforation size on cartilage healing was observed. Nonetheless, Chen and colleagues conducted many investigations assessing the histological features of the newly generated tissue subsequent to chondral defect therapy using diverse repair methods and demonstrated varying levels of penetration into the subchondral bone\u003csup\u003e[\u003cspan additionalcitationids=\"CR27\" citationid=\"CR26\" class=\"CitationRef\"\u003e26\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR28\" class=\"CitationRef\"\u003e28\u003c/span\u003e]\u003c/sup\u003e. Histological findings revealed that the best access to bone marrow was obtained when drilling into deeper areas, as this led to improved defect filling and the production of cartilage with a higher hyaline content\u003csup\u003e[\u003cspan citationid=\"CR27\" class=\"CitationRef\"\u003e27\u003c/span\u003e]\u003c/sup\u003e. In addition, Benthien et al. introduced the nanofracture technique using smaller-diameter and deeper subchondral bone needle perforations to produce cartilage with greater hyaline content\u003csup\u003e[\u003cspan citationid=\"CR36\" class=\"CitationRef\"\u003e36\u003c/span\u003e]\u003c/sup\u003e. Oddly enough, there are no precise guidelines regarding the depth or hole diameter for subchondral drilling.\u003c/p\u003e \u003cp\u003eTo further clarify the roles of microfracture hole diameter and depth in the repair of full-thickness osteochondral defects, we induced full-thickness cartilage defects at two distinct diameters and depths in a preclinical rabbit model. We specifically examined the hypothesis that osteochondral repair is improved when the subchondral bone is perforated with a small-diameter and large-depth device. To rule out other factors that could interfere with the repair process, we applied only one drill hole size per defect and the same number of drill holes for each treatment. We also included a group of defects with simple debridement of subchondral bone to investigate the effect of microfracture itself.\u003c/p\u003e"},{"header":"Materials and Methods","content":"\u003cdiv id=\"Sec3\" class=\"Section2\"\u003e \u003ch2\u003e2.1 Experimental animals\u003c/h2\u003e \u003cp\u003e The use of animals followed the Committee for Animal Use of the 920th Hospital of the PLA Joint Logistics Support Force (Ethics 2022-072 (Department)-01). Sixty healthy New Zealand White rabbits, aged 4 months and weighing approximately 3\u0026thinsp;\u0026plusmn;\u0026thinsp;0.2 kg, with no sex limit, were purchased from Kunming Kejing Co., Ltd. The rabbits were randomly divided into 5 groups (n\u0026thinsp;=\u0026thinsp;12 each): (1) osteochondral defect (Group D); (2) osteochondral defect\u0026thinsp;+\u0026thinsp;diameter 0.4 mm and depth 2 mm MF (Group A1); (3) osteochondral defect\u0026thinsp;+\u0026thinsp;diameter 0.4 mm and depth 9 mm MF (Group A2); (4) osteochondral defect\u0026thinsp;+\u0026thinsp;diameter 0.7 mm and depth 2 mm MF (Group B1); and (5) osteochondral defect\u0026thinsp;+\u0026thinsp;diameter 0.7 mm and depth 9 mm MF (Group B2) (Table \u003cspan refid=\"Tab1\" class=\"InternalRef\"\u003e1\u003c/span\u003e). Before the study began, the animals were kept under standard laboratory conditions.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec4\" class=\"Section2\"\u003e \u003ch2\u003e2.2 Microfracture surgery\u003c/h2\u003e \u003cp\u003eSixty rabbits were randomly selected for the right or left knee joints using a computer and anaesthetized with pentobarbital sodium (3%, 1 mL/kg; Shandong Huamu Pharmaceutical Co., Ltd.). After successful anaesthesia, an osteochondral defect (diameter, 5 mm; depth, 2 mm) was created in the distal femur (trochlear groove) of 1 limb in all 60 rabbits using a trephine drill as previously described\u003csup\u003e[\u003cspan citationid=\"CR37\" class=\"CitationRef\"\u003e37\u003c/span\u003e]\u003c/sup\u003e. Microfracture was performed immediately after the osteochondral defect was created using 0.7 and 0.4 mm drill bits at depths of 2 and 9 mm respectively, with a density of 5 bur holes per 5 mm of osteochondral defect, as previously described\u003csup\u003e[\u003cspan citationid=\"CR30\" class=\"CitationRef\"\u003e30\u003c/span\u003e, \u003cspan citationid=\"CR38\" class=\"CitationRef\"\u003e38\u003c/span\u003e]\u003c/sup\u003e. An osteochondral defect rather than a chondral defect was created, as previous animal studies have shown that when only cartilage is damaged in rabbits, it is possible to for the damage to heal without treatment\u003csup\u003e[\u003cspan citationid=\"CR39\" class=\"CitationRef\"\u003e39\u003c/span\u003e, \u003cspan citationid=\"CR40\" class=\"CitationRef\"\u003e40\u003c/span\u003e]\u003c/sup\u003e. The 2-mm depth of the defect resulted in partial removal of the subchondral cortical bone, and it did not reach the cancellous bone. All procedures were carefully performed, and the tools were cooled with water to prevent heat damage to the cartilage and bone. Care was taken to preserve the miniclots formed on the microfracture holes. Drilling was chosen instead of using an awl because recent literature has shown its superiority to microfracture using an awl. Furthermore, drilling is more reproducible with regard to depth and location, particularly in small-animal models\u003csup\u003e[\u003cspan citationid=\"CR41\" class=\"CitationRef\"\u003e41\u003c/span\u003e]\u003c/sup\u003e. The rabbits were randomly distributed into 5 groups (n\u0026thinsp;=\u0026thinsp;12 per group) as follows: Groups D, A1, A2, B1, and B2 (Table \u003cspan refid=\"Tab1\" class=\"InternalRef\"\u003e1\u003c/span\u003e). The rabbits were fed standard feed and water ad libitum. All rabbits were allowed to walk freely in their cages with no movement restrictions. Veterinary staff from the Animal Research Laboratory of a tertiary hospital provided standard postoperative care for the rabbits, including infection prevention, pain reduction and wound observation. All rabbits were sacrificed at either 6 or 12 weeks after surgery for analysis.\u003c/p\u003e \u003cp\u003e \u003cdiv class=\"gridtable\"\u003e\u003ctable float=\"Yes\" id=\"Tab1\" border=\"1\"\u003e \u003ccaption language=\"En\"\u003e \u003cdiv class=\"CaptionNumber\"\u003eTable 1\u003c/div\u003e \u003cdiv class=\"CaptionContent\"\u003e \u003cp\u003eDescription of Groups\u003c/p\u003e \u003c/div\u003e \u003c/caption\u003e \u003ccolgroup cols=\"3\"\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c1\" colnum=\"1\"\u003e\u003c/div\u003e \u003cdiv align=\"char\" char=\".\" class=\"colspec\" colname=\"c2\" colnum=\"2\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c3\" colnum=\"3\"\u003e\u003c/div\u003e \u003cthead\u003e \u003ctr\u003e \u003cth align=\"left\" colname=\"c1\"\u003e \u003cp\u003eGroups\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c2\"\u003e \u003cp\u003en\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c3\"\u003e \u003cp\u003eProcedure\u003c/p\u003e \u003c/th\u003e \u003c/tr\u003e \u003c/thead\u003e \u003ctbody\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eD group\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e \u003cp\u003e12\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003eosteochondral defect only(5 mm in diameter and 2 mm in depth)\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eA1 group\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e \u003cp\u003e12\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003eosteochondral defect\u0026thinsp;+\u0026thinsp;MF (0.4 mm in diameter and 2 mm in depth)\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eA2 group\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e \u003cp\u003e12\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003eosteochondral defect\u0026thinsp;+\u0026thinsp;MF (0.4 mm in diameter and 9 mm in depth)\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eB1 group\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e \u003cp\u003e12\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003eosteochondral defect\u0026thinsp;+\u0026thinsp;MF (0.7 mm in diameter and 2 mm in depth)\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eB2 group\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e \u003cp\u003e12\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003eosteochondral defect\u0026thinsp;+\u0026thinsp;MF (0.7 mm in diameter and 9 mm in depth)\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003c/tbody\u003e \u003c/colgroup\u003e \u003c/table\u003e\u003c/div\u003e \u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec5\" class=\"Section2\"\u003e \u003ch2\u003e2.3 Macroscopic Analysis\u003c/h2\u003e \u003cp\u003eThe distal femur was immediately dissected after euthanasia as previously described\u003csup\u003e[\u003cspan citationid=\"CR56\" class=\"CitationRef\"\u003e56\u003c/span\u003e]\u003c/sup\u003e. Two orthopaedists and two histologists performed gross observations in a blinded manner, and the results were evaluated using the International Cartilage Repair Society (ICRS) scoring system\u003csup\u003e[\u003cspan citationid=\"CR42\" class=\"CitationRef\"\u003e42\u003c/span\u003e]\u003c/sup\u003e. The ICRS scores were averaged from the separate evaluations. All excised distal femurs were photographed with a digital camera.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec6\" class=\"Section2\"\u003e \u003ch2\u003e2.4 Micro-CT\u003c/h2\u003e \u003cp\u003eTo avoid desiccation during scanning, parafilm was placed over the distal femurs after fixation. A 3-dimensional SkyScan 1276 Micro-CT (Bruker micro-CT, Kontech, Belgium) was used to scan the femurs with the following parameters: voltage, 100 kV; current, 200 \u0026micro;A; AI\u0026thinsp;+\u0026thinsp;Cu filter; voxel size, 20 \u0026micro;m; and rotation step, 0.4\u0026deg;. The images were reconstructed using NRecon software (Bruker micro-CT, Kontech, Belgium) with the following settings: the annular artefact was corrected to 5, and smoothing was applied to 3. In addition, 200 slices (4 mm) in the middle of each defect area were analysed, and the same threshold (cross-sectional view) was utilized to evaluate subchondral bone healing\u003csup\u003e[\u003cspan citationid=\"CR56\" class=\"CitationRef\"\u003e56\u003c/span\u003e]\u003c/sup\u003e.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec7\" class=\"Section2\"\u003e \u003ch2\u003e2.5 Histological Analysis\u003c/h2\u003e \u003cp\u003eAfter decalcification, the tissues were subjected to a series of gradations in alcohol to remove moisture, followed by xylene clearing, processing, and paraffin embedding. Each paraffin block was attached to the cutting device, the slice thickness was set at 4 \u0026micro;m, paraffin slices were made, and water was used to dewax the slices. The sections were subjected to haematoxylin and eosin (G1120, Solarbio, Shanghai, China), Safranin O (G1371, Solarbio, Shanghai, China) and Alcian blue (G1560, Solarbio, Shanghai, China) staining. A fluorescence microscope (Nikon Eclipse E100, Nikon, Japan) was used for microscopic examination, and panoramic scanning was performed using an imaging system (Nikon DS-U3, Nikon, Japan) for image acquisition and analysis. Cartilage repair was assessed through the modified O\u0026rsquo;Driscoll and ICRS grading systems\u003csup\u003e[\u003cspan citationid=\"CR43\" class=\"CitationRef\"\u003e43\u003c/span\u003e]\u003c/sup\u003e based on Alcian blue and Safranin O staining.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec8\" class=\"Section2\"\u003e \u003ch2\u003e2.6 Immunohistochemical Analysis\u003c/h2\u003e \u003cp\u003eImmunohistochemistry was carried out according to previously reported methods\u003csup\u003e[\u003cspan citationid=\"CR56\" class=\"CitationRef\"\u003e56\u003c/span\u003e]\u003c/sup\u003e. The sections were deparaffinized using xylene and gradient ethanol, rehydrated with deionized water, and then incubated for 30 minutes at room temperature with 2% hyaluronidase (H3506-5G; Sigma) in phosphate-buffered saline (pH 7.4) for antigen retrieval. Next, the sections were incubated with anti-type 1 collagen (1:500, 14695-1-AP; Proteintech Group, Wuhan, China) and anti-type 2 collagen (1:500, 28459-1-AP; Proteintech Group, Wuhan, China) overnight, and PBS was used to wash away the residue antibody. Afterwards, the sections were incubated with a goat anti-rabbit secondary antibody (K5007, Dako, Shanghai, China) conjugated with HRP for 50 minutes. Then, diaminobenzidine (DA1016, Solarbio, Shanghai, China) was used to develop the colour, and nuclear counterstaining was performed with haematoxylin. After being dehydrated, the slides were sealed with neutral gum. Finally, a fluorescence microscope (Nikon Eclipse E100, Nikon, Japan) was used to observe the sections, and an imaging system (Nikon DS-U3, Nikon, Japan) was used to perform panoramic scanning and collect the images. The positive ratio of type I collagen to type II collagen was calculated using ImageJ software (version 1.8.0.345) after image acquisition, and the different diameter and depth MMF groups were analysed for significant differences from the blank control group.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec9\" class=\"Section2\"\u003e \u003ch2\u003e2.7 Statistical Analysis\u003c/h2\u003e \u003cp\u003eContinuous variables are presented as the mean\u0026thinsp;\u0026plusmn;\u0026thinsp;standard deviation (SD) and were analysed by IBM SPSS Statistics 26.0 statistical software. The positive area ratios of type I collagen and type II collagen were calculated using ImageJ (version 1.8.0.345). ANOVA was used for statistical analysis, followed by Tukey\u0026rsquo;s post hoc test for two-group comparisons. The data were analysed using GraphPad Prism (version 10.0). A value of P\u0026thinsp;\u0026lt;\u0026thinsp;0.05 was considered statistically significant.\u003c/p\u003e \u003c/div\u003e"},{"header":"Results","content":"\u003cdiv id=\"Sec11\" class=\"Section2\"\u003e \u003ch2\u003e3.1 Micro-CT, gross observation and ICRS score assessment of subchondral bone healing 6 weeks after surgery\u003c/h2\u003e \u003cp\u003eSix weeks postoperatively, gross observation revealed that the cartilage in the defect area in Group A2 had essentially healed, and the colour was nearly the same as that of the surrounding host cartilage. The defect areas were poorly healed in Groups D and A1, with small amounts of regenerated cartilage formed in the defect areas. Groups B1 and B2 exhibited incomplete cartilage healing in the defect areas, though the colour of the regenerated cartilage was essentially the same as that of the surrounding cartilage (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003ec). We performed micro-CT of the damaged bones, and the reconstructed images showed that the subchondral bone defects in Group A2 had essentially healed. However, the subchondral bone damage had not healed in Groups A1, B1, B2 and D, and the healing of the microfracture holes was incomplete (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003ea). A cross-sectional view of the middle area of the defect further showed that subchondral bone healing was greatest in Group A2. Small amounts of bone trabeculae were observed in the subchondral bone in Groups B1 and B2, and Groups D and A1 had more obvious subchondral bone defect areas and no significant bone trabecular regeneration (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eb). Group A2 had the highest ICRS score, as determined by macroscopic evaluation, and the score was significantly greater than that in Group D (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003ed).\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec12\" class=\"Section2\"\u003e \u003ch2\u003e3.2 H\u0026amp;E staining and Alcian blue staining were used to evaluate the effects of cartilage repair\u003c/h2\u003e \u003cp\u003eWe performed histological analyses on the defect areas of the distal femur cartilage. H\u0026amp;E staining revealed very light pink-stained cartilage layers in all groups. In Groups A1, A2, B1 and B2, the microfracture hole areas were thickened, and regenerated cartilage extended to the subchondral bone. The surface of the regenerated cartilage of Group B1 was not stained uniformly. Full cartilaginous covering of the defect (weak staining), which was morphologically very thick in the groove area, was observed in Group A1. Compared with those in Group A1, the cartilage in both Groups A2 and B2 appeared more like hyaline cartilage, demonstrating well-regenerated subchondral bone with pink bone matrix and less thickened cartilage. The surface of the regenerated cartilage of Group B1 was not stained uniformly. However, in Group D, less cartilage had regenerated in the defect area (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003ea). Alcian blue staining showed that the regenerated cartilage areas in Groups A1 and B1 were similar. Both groups displayed hypertrophy and overgrowth, with focal areas of subchondral bone formation, fibrillation, and cysts. In both Groups A2 and B2, the cartilage exhibited normal structural characteristics with relatively thin blue matrix. However, in Group D, no blue cartilage matrix was observed in the defect area, while the growth plate showed robust blue staining, indicating the absence of regenerated cartilage (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eb)\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec13\" class=\"Section2\"\u003e \u003ch2\u003e3.3 Histology Scores Demonstrated Optimal Cartilage Repair in the A2 Group\u003c/h2\u003e \u003cp\u003eWe further performed Safranin O staining for glycosaminoglycans (GAGs), an important structural component of the cartilage matrix. The repaired cartilage in Groups B1 and B2 demonstrated weak Safranin O positivity without full regeneration of the subchondral bone and still showed orange‒red staining. In contrast, Groups A2 and A1 showed robust orange‒red staining at the cartilage surface. In Group D, the defect demonstrated no signs of healing, with only subchondral bone and fibrotic tissues observed at the defect site. The growth plate cartilage was stained orange‒red (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003ea).. On O\u0026rsquo;Driscoll scoring, Group A2 demonstrated a significantly greater score than did Groups A1, B1, B2 and D. Group A1, A2, B1, B2 and D were analysed by ANOVA, and the differences were statistically significant (p\u0026thinsp;\u0026lt;\u0026thinsp;0.001) (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eb).\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec14\" class=\"Section2\"\u003e \u003ch2\u003e3.4 Immunohistochemical Assessments of type 1 collagen (Col-1) and type 2 collagen (Col-2)\u003c/h2\u003e \u003cp\u003eSix weeks after surgery, we examined the expression level of Col-1, which is related to fibrocartilage. The brown indicates collagen I, and at 20\u0026times; magnification, a large amount of collagen I staining was observed on the surface of regenerated cartilage in all MMF groups; high expression was observed in Groups A1, A2, B1 and B2; subchondral bone was not completely healed in all groups; and a small amount of collagen I-positive cartilage was observed in Group D. At 100\u0026times; magnification, fibrosis on the surface of regenerated cartilage-central subchondral bone was observed in all MMF groups, and the fibrosis in Group A2 was less than those in the other groups. The ratio of collagen I-positive cells in Group B1 was the highest, and paired t tests between Group D and Groups A1, A2, B1, and B2 revealed that the values conformed to a normal distribution, and the differences were statistically significant (P\u0026thinsp;\u0026lt;\u0026thinsp;0.001) (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003ea and Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eb).\u003c/p\u003e \u003cp\u003eWe performed immunohistochemistry to measure Col-2. Brown indicates Col-2, and at 20\u0026times; magnification, a small amount of brown collagen II staining was observed on the surface of regenerated cartilage in the MMF groups, and the regenerated cartilage in the cartilage defect area was not completely filled. There was more collagen II-positive cartilage in Group A2 than in the other groups. Subchondral bone staining was intense in Group A2, and it was not completely healed in Group B2. A very small amount of collagen II-positive cartilage was observed in Group D. The regenerated cartilage surfaces in Groups A1 and B2 appeared to be stripped and poorly junctioned, and negative collagen II staining of the cartilage surface and a large number of poor junctions on the cartilage surface were observed in Group D. The positive ratio of collagen II was highest in Group A2, and the paired t test between Group D and Groups A1, A2, B1, and B2 showed that the values conformed to a normal distribution, and the differences were statistically significant (P\u0026thinsp;\u0026lt;\u0026thinsp;0.001) (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003ec and Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003ed).\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec15\" class=\"Section2\"\u003e \u003ch2\u003e3.5 Gross observation and ICRS score assessment of subchondral bone healing 12 weeks after surgery\u003c/h2\u003e \u003cp\u003eAt 12 weeks postoperative, gross observation revealed that the cartilage in the defect areas in the MMF groups had essentially healed, and the colour of Group A2 was basically the same as that of the surrounding host cartilage. The difference in the colour of the regenerated cartilage between Groups A1 and B2 and the surrounding cartilage was greater than that of Group B1, the central point of regenerated cartilage in the defect area of Group B2 had a dotted depression, and the colour of regenerated cartilage in the defect area of Group D had a greater difference in the colour of the regenerated cartilage compared with that of the surrounding host cartilage (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003ea). Group A2 had the highest ICRS score, as determined by macroscopic evaluation, and the score was significantly greater than that in Group D. Analysis of variance revealed that the differences among Groups A1, A2, B1, B2 and D were statistically significant (P\u0026thinsp;\u0026lt;\u0026thinsp;0.001). (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003eb).\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec16\" class=\"Section2\"\u003e \u003ch2\u003e3.6 H\u0026amp;E staining and Safranin O staining to evaluate cartilage repair 12 weeks after surgery\u003c/h2\u003e \u003cp\u003eTwelve weeks after surgery, compared with those in Groups A1, B1 and B2, the regenerated cartilage in Group A2 was thicker, the interface between the new cartilage and host cartilage was more fully integrated, the cartilage and subchondral bone were evenly distributed, and the cartilage was hyaline cartilage-like at 12 weeks after the operation, while one side of the subchondral bone was not completely healed in Group B1, the central subchondral bone was not healed in Group B2, and the central regenerated cartilage was missing in Group D. (Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003ea). In Alcian blue staining, the nucleus is stained red, and the cytoplasm is stained light red. The cartilage layer was not connected continuously, the cartilage surface was obviously fibrotic, a few adipocytes were observed in the cartilage layer, and the defect area also showed light blue cartilage. In Groups A1 and B2, deep blue cartilage was observed from the cartilage layer to the subchondral bone, a small amount of adipocytes was noted in the cartilage layer, and a small amount of fibrosis was observed on the surface of the cartilage. In Group A2, the morphology of regenerated cartilage was normal, and dark blue cartilage was noted from the centre of the cartilage layer to the subchondral bone. In Group D, there was a large amount of fibrosis in the central part of the cartilage defect, a very small amount of dark blue cartilage and a large number of adipocytes in the cartilage layer (Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003eb).\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec17\" class=\"Section2\"\u003e \u003ch2\u003e3.7 Safranin O staining and O\u0026rsquo;Driscoll scores for evaluating cartilage repair 12 weeks after surgery\u003c/h2\u003e \u003cp\u003eAfter 12 weeks, we carried out Safranin O staining. At 20\u0026times; magnification, the middle part of the regenerated cartilage to the subchondral bone in the MF groups were Safranin O positive, and the surface of the cartilage was colourless. Compared with those in Groups A1 and B1, the repaired cartilage showed strong orange‒red staining in Group A2, strong orange‒red staining in Group B2 and no orange‒red staining in the centre of regenerated cartilage in Group D(Fig.\u0026nbsp;\u003cspan refid=\"Fig7\" class=\"InternalRef\"\u003e7\u003c/span\u003ea). The histological O'Driscoll scores of all the samples were evaluated 12 weeks after the operation, and the score in Group A2 was the highest. Groups A1, A2, B1, B2 and D were analysed by ANOVA, and the differences were statistically significant (p\u0026thinsp;\u0026lt;\u0026thinsp;0.001) (Fig.\u0026nbsp;\u003cspan refid=\"Fig7\" class=\"InternalRef\"\u003e7\u003c/span\u003eb).\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec18\" class=\"Section2\"\u003e \u003ch2\u003e3.8 Immunohistochemical assessment of Col-1 and Col-2 12 weeks postoperatively\u003c/h2\u003e \u003cp\u003eLarge amounts of collagen I staining were observed on the surface of regenerated cartilage in the MF groups, while high expression was detected in Groups A1 and B1. The staining ratio in Group A2 at 12 weeks was significantly lower than that at 6 weeks. Subchondral bone had not completely healed in any of the groups, and a very small amount of collagen I-positive cartilage was found in Group D. Fibrosis on the surface of regenerated cartilage-central subchondral bone was observed in all the MF groups, though there was less fibrosis in Group A2 than in the other groups (Fig.\u0026nbsp;\u003cspan refid=\"Fig8\" class=\"InternalRef\"\u003e8\u003c/span\u003ea). The ratio of collagen I-positive cells in Group D was the highest, and paired t tests between Group D and Groups A1, A2, and B1 revealed that the values conformed to a normal distribution, and the differences were statistically significant (P\u0026thinsp;\u0026lt;\u0026thinsp;0.001). (Fig.\u0026nbsp;\u003cspan refid=\"Fig8\" class=\"InternalRef\"\u003e8\u003c/span\u003ea and Fig.\u0026nbsp;\u003cspan refid=\"Fig8\" class=\"InternalRef\"\u003e8\u003c/span\u003eb).\u003c/p\u003e \u003cp\u003eIn type II collagen staining, brown indicates collagen II. A small amount of brown collagen II staining was observed on the surface of the regenerated cartilage in each of the MMF groups, and the regenerated cartilage in the cartilage defect areas was almost completely filled. Compared with Groups A1, B1 and B2, there was more collagen II-positive cartilage in Group A2, but subchondral bone was strongly stained and did not heal completely in Groups A2 and B2, and there was very little collagen II-positive cartilage in Group D. Among the MMF groups, collagen II staining in the cartilage region was evident in Group A2, which had tightly connected cartilage surfaces and a small number of cracks on the cartilage surfaces; the regenerated cartilage surfaces were not tightly connected in Groups A1 and B2; and negative collagen II staining of the cartilage surfaces and a large number of cracks on the cartilage surfaces were observed in Group D (Fig.\u0026nbsp;\u003cspan refid=\"Fig8\" class=\"InternalRef\"\u003e8\u003c/span\u003ec). The positive ratio of collagen II in Group A2 was the highest, and the paired t test between Group D and Groups A1 and A2 revealed that the values conformed to a normal distribution, and the differences were statistically significant (P\u0026thinsp;\u0026lt;\u0026thinsp;0.001) (Fig.\u0026nbsp;\u003cspan refid=\"Fig8\" class=\"InternalRef\"\u003e8\u003c/span\u003ec and Fig.\u0026nbsp;\u003cspan refid=\"Fig8\" class=\"InternalRef\"\u003e8\u003c/span\u003ed).\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003c/div\u003e"},{"header":"Discussion","content":"\u003cp\u003eThe primary finding of the present study was that 0.4 mm-diameter and 9 mm-deep microfracture drill holes significantly improved articular cartilage repair compared to drill holes of larger diameter and smaller depth. Second, compared with control defects that were debrided alone, microfracture treatment involving the use of either drill holes of different diameters or depths led to better histological cartilage repair. The application of microfracture as a treatment option for cartilage abnormalities has been thoroughly examined in the literature, and various perforation techniques have been investigated\u003csup\u003e[\u003cspan additionalcitationids=\"CR27 CR28\" citationid=\"CR26\" class=\"CitationRef\"\u003e26\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR29\" class=\"CitationRef\"\u003e29\u003c/span\u003e]\u003c/sup\u003e. However, the effects of different perforation diameters and depths on cartilage healing have not been well studied. In this experimental study, microfractures (subchondral perforations) from osteochondral defects of different diameters and depths were compared macroscopically, histologically, and immunohistochemically. The main findings of this study were that microfractures with different diameters and depths demonstrated different healing characteristics. The results showed that a diameter of 0.4 mm and a depth of 9 mm could significantly improve the repair of rabbit knee cartilage. Moreover, the content of hyaline cartilage in the repaired tissue was greater than those in the other groups.\u003c/p\u003e \u003cp\u003eAs a result, considerable effort has been made to develop and modify microfracture. Although MF has become the main option for therapy for cartilage lesions owing to its technical simplicity, minimal invasiveness, limited availability, low cost, and autogenous nature, which enhance cartilage repair by releasing BMSCs through small drill holes in cartilage defects\u003csup\u003e[\u003cspan citationid=\"CR38\" class=\"CitationRef\"\u003e38\u003c/span\u003e, \u003cspan citationid=\"CR44\" class=\"CitationRef\"\u003e44\u003c/span\u003e]\u003c/sup\u003e, the long-term efficacy of MF was weak in many follow-up examinations due to the production of more fibrocartilage after the procedure\u003csup\u003e[\u003cspan additionalcitationids=\"CR45\" citationid=\"CR44\" class=\"CitationRef\"\u003e44\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR46\" class=\"CitationRef\"\u003e46\u003c/span\u003e]\u003c/sup\u003e. In addition, several alternative treatment modalities have emerged to address focal chondral defects that could promote hyaline cartilage repair, including particulate cartilage allografts\u003csup\u003e[\u003cspan citationid=\"CR47\" class=\"CitationRef\"\u003e47\u003c/span\u003e, \u003cspan citationid=\"CR48\" class=\"CitationRef\"\u003e48\u003c/span\u003e]\u003c/sup\u003e, osteochondral allografts or autografts\u003csup\u003e[\u003cspan citationid=\"CR49\" class=\"CitationRef\"\u003e49\u003c/span\u003e]\u003c/sup\u003e, and hyaline-like cartilage such as autologous chondrocyte implantation\u003csup\u003e[\u003cspan citationid=\"CR50\" class=\"CitationRef\"\u003e50\u003c/span\u003e]\u003c/sup\u003e. Microfracture techniques to alter bone marrow stimulation for the purpose of regenerating cartilage similar to normal hyaline cartilage, and different cartilage repair effects have been shown by varying the diameter, depth, number of drilled holes, spacing of holes, location, and other characteristics of microfractures alone\u003csup\u003e[\u003cspan citationid=\"CR27\" class=\"CitationRef\"\u003e27\u003c/span\u003e, \u003cspan citationid=\"CR29\" class=\"CitationRef\"\u003e29\u003c/span\u003e, \u003cspan citationid=\"CR33\" class=\"CitationRef\"\u003e33\u003c/span\u003e, \u003cspan additionalcitationids=\"CR52\" citationid=\"CR51\" class=\"CitationRef\"\u003e51\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR53\" class=\"CitationRef\"\u003e53\u003c/span\u003e]\u003c/sup\u003e. However, several of these modified microfracture approaches have yet to be utilized in clinical. Interestingly, the effects of perforations of different diameters and depths on osteochondral healing have received little research attention.\u003c/p\u003e \u003cp\u003eFew studies have examined the impacts of microfracture drill hole diameter and depth on osteochondral repair. In a small animal model, no differences were reported when full-thickness cartilage defects in the trochlea were subjected to drilling using either 0.5-mm (proximal trochlea) or 0.9-mm (distal trochlea) holes at 6 months postoperatively\u003csup\u003e[\u003cspan citationid=\"CR32\" class=\"CitationRef\"\u003e32\u003c/span\u003e]\u003c/sup\u003e. The application of 1.0-mm subchondral drill holes significantly improved histological articular cartilage repair compared to 1.8-mm holes, as indicated by their better average total score. Moreover, matrix staining for Safranin O was significantly increased, suggesting that the repair tissue in the 1.0-mm drill holes contained more proteoglycans. In addition, the number of cells resembling articular chondrocytes within the repair of 1.0-mm drill holes was also greater, indicating that the structure of the repaired tissue more closely resembled that of cartilage.\u003c/p\u003e \u003cp\u003eIn good agreement with these findings, the immunoreactivity to type II collagen, an essential component of the normal cartilage extracellular matrix, was considerably greater in the tissue repaired with 1.0-mm drill holes. Conversely, the immunoreactivity to type I collagen (an indication of fibrocartilaginous repair) was considerably lower in the tissue repaired with 1.0-mm drill holes than in that repaired with 1.8-mm drill holes\u003csup\u003e[\u003cspan citationid=\"CR29\" class=\"CitationRef\"\u003e29\u003c/span\u003e]\u003c/sup\u003e. Min and colleagues\u003csup\u003e[\u003cspan citationid=\"CR34\" class=\"CitationRef\"\u003e34\u003c/span\u003e]\u003c/sup\u003e reported that larger holes (diameter of 1.5 mm) increase the access of BMSCs and growth factors to articular cartilage lesions in rabbit knee joints compared with smaller holes (0.8 mm). To date, only one study has investigated the influence of microfracture hole parameters on articular cartilage repair in vivo: Kok et al.\u003csup\u003e[\u003cspan citationid=\"CR35\" class=\"CitationRef\"\u003e35\u003c/span\u003e]\u003c/sup\u003e treated talar osteochondral lesions in a caprine model using microfracture holes 0.45 or 1.10 mm in diameter and 2 or 4 mm in depth. No significant effect of perforation size on cartilage repair was noted after 4 months. However, the applicability of the findings to the present research is limited due to significant differences between the ankle and knee joints\u003csup\u003e[\u003cspan citationid=\"CR54\" class=\"CitationRef\"\u003e54\u003c/span\u003e]\u003c/sup\u003e. Another investigation demonstrated significantly greater quantities of DNA (up to 3.9-fold), proteoglycans (up to 4.2-fold), and type II collagen (up to 4.0-fold) in cartilaginous repair tissue following small-diameter (1.0 mm) subchondral drilling than after large-diameter (1.8 mm) drilling\u003csup\u003e[\u003cspan citationid=\"CR51\" class=\"CitationRef\"\u003e51\u003c/span\u003e]\u003c/sup\u003e. This is consistent with our experimental results. Compared with those in the other groups, the amount of type I collagen in the 0.4 mm diameter sample was much lower, although the amount of type II collagen was significantly greater, demonstrating that an MF with a diameter of 0.4 mm repaired cartilage injuries to more closely resemble normal articular cartilage. Moreover, we discovered that the type I collagen contents were lowest in the defect group at 6 weeks after surgery and highest at 12 weeks after surgery, primarily due to the small amount of cartilage regeneration observed in the defect group at 6 weeks after surgery and the gradual regeneration of cartilage over time after 6 weeks. At 12 weeks, we observed a moderate amount of cartilage regeneration by gross observation and histology. Immunohistochemistry revealed that the regenerated cartilage in the defect group was predominantly type I collagen, which is consistent with the findings of osteochondral defect models established in previous studies\u003csup\u003e[\u003cspan citationid=\"CR38\" class=\"CitationRef\"\u003e38\u003c/span\u003e]\u003c/sup\u003e.\u003c/p\u003e \u003cp\u003eThe repair of articular cartilage injury by MF mainly depends on the recruitment of BMSCs\u003csup\u003e[\u003cspan citationid=\"CR39\" class=\"CitationRef\"\u003e39\u003c/span\u003e]\u003c/sup\u003e. Therefore, providing access to the bone marrow stroma is the first step in BMSC recruitment. Zedde et al.\u003csup\u003e[\u003cspan citationid=\"CR30\" class=\"CitationRef\"\u003e30\u003c/span\u003e]\u003c/sup\u003e conducted microfracture and nanomicrofracture in the sheep knee joint and discovered that nanomicrofracture promoted cartilage regeneration. Additionally, it has been observed that deeper (6 mm) subchondral drilling results in more hyaline cartilage repair than shallower (2 mm) drilling. This is evidenced by increased levels of glycosaminoglycan, type II collagen content, and cartilage defect filling, as well as a decrease in type I collagen\u003csup\u003e[\u003cspan citationid=\"CR27\" class=\"CitationRef\"\u003e27\u003c/span\u003e]\u003c/sup\u003e, which was more consistent with MF cartilage repair at a depth of 9 mm than at a depth of 2 mm in this study. Our findings suggest that microfracture with a diameter of 0.4 mm and a depth of 9 mm can considerably increase rabbit knee cartilage healing, possibly because the hole diameter and depth activate more BMSCs\u003csup\u003e[\u003cspan citationid=\"CR51\" class=\"CitationRef\"\u003e51\u003c/span\u003e]\u003c/sup\u003e.\u003c/p\u003e \u003cp\u003eReconstruction of the subchondral bone is essential for osteochondral repair\u003csup\u003e[\u003cspan citationid=\"CR55\" class=\"CitationRef\"\u003e55\u003c/span\u003e]\u003c/sup\u003e. Compared to 1.8-mm drilling, 1.0-mm drilling had normal bone density of the subchondral bone plate and subarticular cancellous bone, which was very comparable to that of the adjacent subchondral bone, and 1.0-mm drilling contributed to significantly better healing of the subchondral bone plate and a substantial decrease in subchondral bone plate thickening\u003csup\u003e[\u003cspan citationid=\"CR33\" class=\"CitationRef\"\u003e33\u003c/span\u003e]\u003c/sup\u003e. In our study, a drilling diameter of 0.4 mm caused less damage to the subchondral bone and led to better subchondral bone reconstruction and higher quality cartilage.\u003c/p\u003e \u003cp\u003eIn this study, we confirmed that different diameters and depths of MF had positive effects on cartilage repair and that MF with a diameter of 0.4 mm and a depth of 9 mm promoted the expression of type 2 collagen and the regeneration of hyaline cartilage. However, these parameters were chosen by considering previous studies to allow for legitimate comparisons\u003csup\u003e[\u003cspan citationid=\"CR27\" class=\"CitationRef\"\u003e27\u003c/span\u003e, \u003cspan citationid=\"CR29\" class=\"CitationRef\"\u003e29\u003c/span\u003e, \u003cspan citationid=\"CR34\" class=\"CitationRef\"\u003e34\u003c/span\u003e, \u003cspan citationid=\"CR36\" class=\"CitationRef\"\u003e36\u003c/span\u003e, \u003cspan citationid=\"CR56\" class=\"CitationRef\"\u003e56\u003c/span\u003e]\u003c/sup\u003e, which resulted in considerable differences between groups. Our research findings will motivate clinicians to explore viable therapeutic approaches for different diameters and depths of MF to enhance cartilage regeneration, addressing current clinical bottlenecks such as fibrocartilage formation and poor long-term outcomes after MF.\u003c/p\u003e \u003cp\u003eWe recognize that the current study is not without limitations. First, the 12-week follow-up period may be too short to provide enough clinical relevance. Second, the osteochondral defect area in the rabbit knees was created acutely rather than chronically. This makes the cartilage defect model created in animals less similar to articular cartilage damage in humans. Third, we only observed the condition of the subchondral bone by micro-CT at 6 weeks after surgery. Fourth, we did not measure the amount of BMSCs released after microfracture surgery. In addition, while the results from a rabbit osteochondral defect model are intriguing, further research in large animal models and eventually in humans should be conducted before clinical application. Finally, we did not perform a biomechanical evaluation of the regenerated cartilage tissue, but this may constitute future research.\u003c/p\u003e"},{"header":"Conclusion","content":"\u003cp\u003eMMF treatment significantly enhances cartilage repair, with the parameters of 0.4 mm in diameter and 9 mm in depth exhibiting the highest therapeutic efficacy.\u003c/p\u003e"},{"header":"Declarations","content":"\u003cp\u003e\u003cstrong\u003eAcknowledgments\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThis study is financed by Yunnan Science and Technology Department Key Projects(202401AY070001-045、202501AS070164) and Yunnan Orthopedics and Sports Rehabilitation Clinical Medicine Research Center (202102AA310068).\u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAuthors\u003c/strong\u003e\u003cstrong\u003e\u0026rsquo;\u003c/strong\u003e\u003cstrong\u003e\u0026nbsp;Contributions\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eH. T. and T.Z. conceived the study. Z.C. and Z.Y. performed all animal models. Y.X. performed CT analysis. N.Y. ,H.F. and Y.C. performed all histological tests. Z.Y. and Y.Z. wrote the paper. All authors have read and approved final version.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eFunding Declaration\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe authors disclose receipt of the following financial or material support for the research, authorship, and/or publication of this article: this work was supported by Yunnan Science and Technology Department Key Projects(202401AY070001-045、202501AS070164) and Yunnan Orthopedics and Sports Rehabilitation Clinical Medicine Research Center (202102AA310068).\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eData Availability\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eAll datasets presented in this study are included in the article.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eConflicts of Interest\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe authors declare that they have no competing interests.\u003c/p\u003e"},{"header":"References","content":"\u003col\u003e\n\u003cli\u003eMithoefer K, McAdams T, Williams R J, Kreuz, P C, Mandelbaum, B R. 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Sci Rep, 2024,14(1):3811.\u003c/li\u003e\n\u003c/ol\u003e"}],"fulltextSource":"","fullText":"","funders":[],"hasAdminPriorityOnWorkflow":false,"hasManuscriptDocX":true,"hasOptedInToPreprint":true,"hasPassedJournalQc":"","hasAnyPriority":false,"hideJournal":true,"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":"researchsquare","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":true,"externalIdentity":"","sideBox":"","snPcode":"","submissionUrl":"/submission","title":"Research Square","twitterHandle":"researchsquare","acdcEnabled":true,"dfaEnabled":false,"editorialSystem":"","reportingPortfolio":"","inReviewEnabled":false,"inReviewRevisionsEnabled":true},"keywords":"Microfracture, Osteochondral defect, Cartilage repair, Diameter, Depth","lastPublishedDoi":"10.21203/rs.3.rs-5532606/v1","lastPublishedDoiUrl":"https://doi.org/10.21203/rs.3.rs-5532606/v1","license":{"name":"CC BY 4.0","url":"https://creativecommons.org/licenses/by/4.0/"},"manuscriptAbstract":"\u003cp\u003ePrevious studies have confirmed that small-diameter microfracture improves articular cartilage repair more effectively than do large-diameter microfracture and that drilling more deeply improves repair tissue quality. However, in microfracture (MF) surgery, the optimal diameters and depths of the holes drilled into the subchondral bone and their influence on cartilage healing are currently unknown. This study established a rabbit osteochondral defect model and treated cartilage lesions with modified microfracture (MMF) applications of different diameters and depths. Cartilage repair was detected through gross observation, histological analyse and immunohistochemical analyse. The results showed that MF with a diameter of 0.4 mm and a depth of 9 mm enhanced cartilage regeneration at 6 weeks after creating an osteochondral defect and resulted in virtually normal cartilage healing at 12 weeks. The repaired cartilage in the MMF group (diameter 0.4 mm and depth 9 mm) was more hyaline-like than those in the defect and other microfracture groups. In summary, this study confirmed that small-diameter and large-depth MMF could promote cartilage repair.\u003c/p\u003e","manuscriptTitle":"Small-Diameter and Large-Depth Microfracture Improves Cartilage Repair in the Rabbit Osteochondral Defect Model","msid":"","msnumber":"","nonDraftVersions":[{"code":1,"date":"2025-04-21 05:34:58","doi":"10.21203/rs.3.rs-5532606/v1","editorialEvents":[{"type":"communityComments","content":0}],"status":"published","journal":{"display":true,"email":"[email protected]","identity":"researchsquare","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":true,"externalIdentity":"","sideBox":"","snPcode":"","submissionUrl":"/submission","title":"Research Square","twitterHandle":"researchsquare","acdcEnabled":true,"dfaEnabled":false,"editorialSystem":"","reportingPortfolio":"","inReviewEnabled":false,"inReviewRevisionsEnabled":true}}],"origin":"","ownerIdentity":"9c9dabbe-a03a-4c18-ac4a-10261b084bc1","owner":[],"postedDate":"April 21st, 2025","published":true,"recentEditorialEvents":[],"rejectedJournal":[],"revision":"","amendment":"","status":"posted","subjectAreas":[],"tags":[],"updatedAt":"2025-04-27T14:23:24+00:00","versionOfRecord":[],"versionCreatedAt":"2025-04-21 05:34:58","video":"","vorDoi":"","vorDoiUrl":"","workflowStages":[]},"version":"v1","identity":"rs-5532606","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-5532606","identity":"rs-5532606","version":["v1"]},"buildId":"8U1c8b4HqxoKbykW_rLl7","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}