Augmentation of peripheral nerve regeneration by hypoxic allogenic Schwann-like cells in acute nerve injury of Rattus norvegicus

Augmentation of peripheral nerve regeneration by hypoxic allogenic Schwann-like cells in acute nerve injury of Rattus norvegicus | 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 Augmentation of peripheral nerve regeneration by hypoxic allogenic Schwann-like cells in acute nerve injury of Rattus norvegicus Tito Sumarwoto, Romaniyanto Romaniyanto, Heri Suroto, Dwikora Novembri Utomo, and 8 more This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-8423634/v1 This work is licensed under a CC BY 4.0 License Status: Under Review Version 1 posted 9 You are reading this latest preprint version Abstract Background: This study investigates nerve regeneration augmentation using hypoxic allogeneic Schwann-like cells by analyzing HIF-1α, CD-31, Neu-N, α-SMA, NCAM, TGF-β, VEGF, and motor function. Methods : This in-vivo study on Rattus norvegicus Wistar divided subjects into intervention (suture plus hypoxic allogeneic SLCs) and control (suture only) groups. SLCs were derived from Adipose Mesenchymal Stem Cells using Kingham's protocol with 10% PRP and 1% hypoxia. ELISA, IHC, rt-PCR were done at weeks 3 and 6, and walking track analysis with sciatic function index (SFI) was performed from weeks 0 to 6. Results: The intervention group expressed HIF-1αmore clearly, especially in week 6. In addition, there were statistically significant differences in TGF-b(p=0.002), α-SMA (p=0.000), NCAM (p=0.000), and Neu-N (p=0.049) at week 3, as well as TGF-b (p=0.000), α-SMA (p=0.003), CD-31 (p=0.000), NCAM (p=0.000), and Neu-N (p=0.000) at week 6 between interventions and control group. Significant differences were also found in TGF-b, α-SMA, CD-31, NCAM, and Neu-N between weeks 3 and 6 in the intervention group. Furthermore, differences were also found in the sciatic function index at weeks 2 to 6 (p<0.050) between the intervention group and the control group. Conclusion: Administration of hypoxic-conditioned allogeneic SLCs accelerated peripheral nerve regeneration in acute peripheral nerve injury (PNI), as evidenced by increased TGF-blevels, HIF-1α and NCAM expression, the axonal density of peripheral nerves through the expression of NeuN protein, and the number of capillaries through expression of CD-31; decreased expression of α-SMA; and improved motor function. Hypoxic conditioned Schwann-like cells (SLCs) Peripheral nerve regeneration Acute peripheral nerve injury (PNI) Adipose-derived mesenchymal stem cells (AdMSCs) Secretome In-vivo rat model Figures Figure 1 Figure 2 Figure 3 Figure 4 Figure 5 Significance Statement Peripheral nerve injuries often cause lasting disability due to poor regeneration and surgical results. This study shows that transplanting hypoxic-conditioned allogeneic Schwann-like cells (SLCs) significantly enhances nerve repair in acute injuries. Hypoxic preconditioning increased key regenerative factors—HIF-1α, TGF-β, NCAM, Neu-N, and CD-31—and decreased α-SMA, leading to higher axon density, better blood vessel growth, and improved motor recovery. These findings indicate that hypoxic-conditioned SLCs and their secretions are promising, scalable options for faster nerve healing, potentially serving as effective adjuncts to microsurgery and improving patient outcomes. Introduction Peripheral nerve injury (PNI) may cause permanent sensory dysfunction and severe motor impairment. The consequences of these injuries are often severe, affecting daily living and even devastating.(1) Annually, PNI caused by accidents and trauma is 200,000 in the United States and 300,000 in Europe.(2,3) A retrospective study in Surabaya, Indonesia, also showed that brachial plexus injuries were prevalent, especially in men and the 21-30 age group.(4) Another study conducted in Surakarta, Indonesia, also provides a similar description, with the most common etiology being traffic accidents, especially motorcycle-related ones.(5) Traumatic peripheral nerve injury (PNI) remains difficult to manage despite its high prevalence. The current gold standard-nerve grafting with epineural microsuturing—requires advanced skill yet often yields suboptimal outcomes due to inflammation, scar formation, axonal misdirection, and slow regeneration.(6–8) New strategies are needed to accelerate axonal regeneration and functional recovery. Cell-based augmentation therapy offers promise by creating a supportive microenvironment forrepair.(9,10) Among candidate cells, mesenchymal stem cells (MSCs)-notably bone marrow- (BM-MSCs) and adipose-derived (AdMSCs)-stand out for their self-renewal, multipotency, and paracrine effects.(11,12) Schwann cells remain the natural choice for nerve repair but are limited by donor-site morbidity, slow growth, and difficulty in culture.(13,14) AdMSCs can be induced into Schwann-like cells (SLCs), expressing Schwann cell markers and promoting neurite extension, myelination, and neuronal survival via neurotrophic factor secretion and recruitment of endogenous Schwann cells.(15,16) Hypoxic preconditioning (HPC), which cultures cells under 1–5% O₂ before transplantation, mimics the injured tissue milieu and accelerates survival, angiogenesis, and neuroprotection through HIF-1α–mediated pathways, generating a more robust regenerative phenotype.(17,18) The secretome-the spectrum of bioactive molecules released by hypoxia-conditioned SLCs-plays a pivotal role in modulating the injury microenvironment, accelerating axonal regrowth, and supporting functional recovery after PNI.(19,20) This study aims to investigate the augmentation of nerve regeneration after administration of hypoxic conditioned allogeneic SLCs and its secretome in peripheral nerve reinnervation by analyzing several markers, including the expression of HIF-1α, CD-31, and Neu-N protein, α-SMA, and NCAM; TGF-b and VEGF levels; and improvement of motor function with walking track analysis. Materials & Methods Study design This in-vivo experimental study uses white rats ( Rattus norvegicus ) Wistar strain, obtained from the Animal and Stem Cell Research Institute of the Tropical Disease Laboratory, Airlangga University, Surabaya, East Java, Indonesia. Isolation and culture of AdMSCs, preparation of hypoxic SLCs, surgery process (including denervation and reinnervation), and administration of hypoxic SLCs were also carried out there. The process of tissue preparations, immunohistochemical staining, observing micrografts, collecting data, and validating observational data was carried out at the Animal and Stem Cell Research Institute of Tropical Disease Laboratory, Airlangga University, Surabaya, East Java, Indonesia, as well as the Anatomy-Histology Laboratory, and the Anatomical Pathology Laboratory, Faculty of Medicine, Public Health, and Nursing, Gadjah Mada University, Yogyakarta, Indonesia, conducted from May to July 2022. This study was conducted in accordance with the Animal in Research: Reporting In Vivo Experiments (ARRIVE) 2.0 guidelines, and a completed checklist has been submitted as supplementary material.(21) The ethical assessment of this research was considered by the Animal Care and Use Committee (ACUC), Faculty of Veterinary Medicine, Airlangga University, Surabaya, East Java, Indonesia, and received an ethical clearance with a letter-number of 2.KE.038.05.2020 on May 13, 2020. Animal model This study use the white male rat Rattus norvegicus Wistar strain with aged 3-4 months, weighing 200-300 grams (to make the sample more homogeneous), and healthy. The health status was analyzed by examination to active movements, no skin wounds, no red eyes, respiratory rate <60 times per minute, and rectal temperature < 40°C, which was confirmed by a competent veterinarian. The number of replicated experimental is rounded 6 units, determined the sample size formula by Lemeschow (22) , with calculating correction factor. The drop-out criteria in this study were animals that died during the research process. Subsequently, this study divided the research subjects into three groups, one group is control and two groups were intervention group. The control group received denervation and reinnervation with sutures. The intervention group received the denervation and reinnervation group with sutures plus allogeneic SLCs under hypoxic conditions, and their secretome. Animals were randomly allocated to control and intervention groups using a computer-generated random sequence to minimise allocation bias. Blinding was applied throughout the study: the personnel conducting the experiment, outcome assessment, and data analysis were unaware of group allocations. Animal preparation Twenty-four Rattus norvegicus were placed in standard cages (measuring 40x60x15cm) made of plastic trays covered with husks and woven wire and had partitions so that the animals could not mix. The housing density was maintained at two rats per cage to ensure sufficient space and reduce competition for resources. Environmental enrichment included nesting materials and chew toys to promote natural behaviors and improve animal welfare. The cage is hygienic and shielded from wind, rain, and direct sunlight while maintaining an ambient temperature of approximately 15-20°C. Furthermore, the cages were given sufficient lighting with light-dark cycles per 12 hours at 24°C and 50% humidity and maintained under standard laboratory conditions with routine veterinary oversight. Housing was designed to provide both physical and psychological comfort, with regular cleaning schedules and monitoring to prevent overcrowding and ensure a stress-free environment. Experimental animals were also given food and drink ad libitum . The rat meal extract contains 20-25% protein, 5% fat, 40-50% starch, and 5% crude fiber. Each day, 12-20 grams of food and 80-100 cc/kg water was administered. No animals/data were excluded from the analysis. Anesthetic, euthanasia, and post-experimental procedures All invasive procedures, including surgeries and sample collections, were performed under general anesthesia to minimize pain and distress. Rats were anesthetized using an intramuscular injection of 50-55 mg/kg Ketamine hydrochloride (@Ket-A-100) and 25 mg/kg Xylazine hydrochloride (@Xyla 20mg). The depth of anesthesia was confirmed by the absence of reflex responses to stimuli. Post-operative analgesia was included in the experimental protocol to minimize pain and support animal welfare, where feasible. Following surgery, animals received intramuscular injections of Phenylbutazone at a dose of 20 mg/kg once daily for up to three days, based on clinical assessment and antibiotics Enrofloxacin 20-40mg/kg before surgey and for 2 days after surgery as prophylaxis. Animals were monitored daily for signs of pain or distress, and additional analgesic doses were administered if necessary. Subsequently, animals used for blood and tissue sampling were euthanized after the completion of sample collection to prevent post-surgical discomfort. Last, After completion of the experimental procedures, all surviving rats were humanely euthanized with a lethal dose of sodium pentobarbital administered via the carotid artery under deep anesthesia. This method was selected to ensure a rapid and painless death in accordance with AVMA guidelines on euthanasia. This also prevents prolonged captivity and ensures no unnecessary suffering. No animals remained alive post-experiment, adhering to ethical guidelines for the humane treatment of laboratory animals. Preparation of stromal vascular fraction, isolation and culture of AdMSCs Two experimental animals Rattus norvegicus were used to isolate AdMSCs. Animals were kept in a controlled environment. The rats were euthanized, and the abdominal fat pad was extracted. Adipose-derived mesenchymal stem cells (AdMSCs) were isolated using the methodology described by Zuk et al. (2001) and Gimble et al. (2007).(23,24) Subsequently, to verify the cellular identity of cultivated AdMSCs cells, immunofluorescence labeling was performed on AdMSCs from passage 3 using CD14, CD29, CD34, CD44, CD45, CD73, CD90, and CD105 markers. (25,26) Preparation of platelet-rich plasma Blood was collected from three anesthetized rats via the carotid artery. Following blood collection, the animals were euthanized. 40 mL of blood was immediately mixed with citrate phosphate dextrose (CPD) buffer as an anticoagulant, using a 1:5 ratio (1 mL CPD to 5 mL blood), with a CPD concentration of 0.15 mg/mL. The blood sample was centrifuged at a force of 500 g for 5 minutes using the Heraeus Cryfuge 6000i centrifuge from Kendro Laboratory Products in Hessen, Germany. Subsequently, the liquid portion with a high concentration of platelets, known as platelet-rich plasma (PRP), was subjected to a second round of centrifugation at a force of 2000 times the acceleration due to gravity (2000g) for 5 minutes. Subsequently, the platelet pellets from the second spin were resuspended with the supernatant until a final concentration of 1.5 × 10¹² platelets/L was achieved.(27) PRP is needed for the preparation of Schwann-like cells. Preparation of Schwann-like cells (neuronal/ glial trans-differentiation) The Kingham procedure was used to prepare SLCs produced from AdMSC rats, and a 10% PRP solution was administered. (28) During the third pass, the AdMSCs cells were transformed into a phenotype similar to SLC-dAdMSCs through two stages. Initially, the culture medium was replaced with a medium containing 1 mM³ - mercaptoethanol (Scharlau Chemicals) for 24 hours. This was followed by treatment with 35 ng/mL all-trans retinoic acid (Sigma-Aldrich) for 72 hours. Subsequently, the cells were exposed to a differentiation medium containing 5 ng/mL PDGF, 10 ng/mL bFGF (both from PeproTech), 14 µM forskolin (Sigma-Aldrich), 252 ng/mL neuregulin-1 (R&D Systems), and 10% PRP. This differentiation was maintained for at least 14 days before the cells were characterized. (28,29) Cells used in this study were from passages 3 to 6. Oxygen levels during culture were maintained at 21% using the ProOx-C-chamber 110 (Biospherix, Redfield, NY) under tightly controlled conditions for 24 hours. (30) Isolation of rat sciatic nerve-derived stem cells Segments of sciatic nerve approximately 1 cm were dissected and cleared of the epineurium. The tissue was then cut into ~1 mm pieces and enzymatically digested using 2 mg/mL collagenase NB4. Then, dispersion was carried out in DMEM/F-12 for 15 minutes at 37°C, followed by centrifugation and resuspension in DMEM/F-12, which contained 10% FBS. Subsequently, the cells obtained were cultured at 37°C with 5% CO 2 atmosphere. After 48 hours, the supernatant was discarded, and the remaining cells were treated with 1 mg/mL collagenase NB4 to separate them from the fibroblast cells. Hypoxic pre-condition treatment on Schwann-like cells Hypoxic preconditioning treatment was performed on SLCs and rat sciatic nerve-derived SCs with O 2 levels of 1%, 3%, and 5% in preliminary studies. (31) Hypoxic preconditioning is carried out by stimulating in a water-saturated gas mixture at 1%, 3% or 5% of O 2 , 5% of CO 2 , and 94% N 2 at 37°C for 24 hours. The levels of several growth factors were examined, including BDNF, GDNF, bFGF, NGF, TGF-b, and VEGF, using the ELISA from SLCs and SCs, respectively, in 21% normoxia and 1%, 3%, and 5% hypoxic conditions, which result that no significant differences in growth factor secretion were observed between SLCs and SCs. Furthermore, growth factor concentrations were observed in SLCs for all oxygen concentrations, and the result was that 1% oxygen content had the highest growth factor concentration. This research has been carried out in previous studies, (31) so that SLCs with 1% hypoxic conditions were used for this study. The 1% hypoxic preconditioning of SLC-dAdMSCs was performed in a controlled ProOx-C-chamber system (ProOx 110; Biospherix, Redfield, NY) for 24 hours. (30) Secretome production from cell cultures under h ypoxic conditions Passage/sub-culture of cells that have reached 70-80% confluence for propagation. Re-seeding or re-planting the cell culture on a 100 mm petri dish with a density of 5x10 5 cells per 100 mm petri dish. Incubate the grown cells in a 1% hypoxic incubator for 4 days. Collect the supernatant/medium from the petri dish and collect it in a connical tube. Centrifuge at 3000 rpm for 5 minutes. Filter the supernatant using a 0.45 µm pore filter and collect it in a new connical tube. Store the supernatant (secretome) at -20 o C Denervation and reinnervation procedure All surgical procedures were performed under anesthesia to ensure humane treatment of the animals. In the control group, the sciatic nerve was denervated by cutting the sciatic nerve (neurectomy), which was opened through the gluteal region of the leg and then reinnervated with sutures using 10-0 nylon thread after waiting for 10 minutes by giving the amniotic membrane to the nerve reinnervation area. In the two intervention groups, the sciatic nerve was denervated, then reinnervated with sutures using 10-0 nylon thread and given allogeneic SLCs under 1% hypoxic conditions, and one group another with secretome of allogeneic SLCs under 1% hypoxic conditions. The amnion membrane was used as a scaffold in the nerve reinnervation area. The technique of joining the proximal to the distal nerve section was performed using microsurgery with four interrupted sutures on the epineural using 10-0 polypropylene monofilament. Animal dissection and nerve extraction Experimental animals were anesthetized using intramuscular injection of 50-55 mg/kg Ketamine hydrochloride (@Ket-A-100) and 25mg/kg xylazine hydrochloride (@Xyla 20mg). After skin disinfection, the hair was scraped off with a 10% povidone-iodine solution. The right sciatic nerve was accessed via a skin incision and carefully isolated using a gluteal muscle-splitting approach. The left leg served as an internal control. The sciatic nerve axonotomesis procedure in the mid-hind legs of rats by Savastano (2014) was used in this study. (32) All surgery processes were performed by the same investigator, using microsurgery loops and tools. Enzyme-linked immunosorbent assay (ELISA) examination To assess the serum levels of TGF-b and VEGF, enzyme-linked immunosorbent assay (ELISA) was performed. The procedure began with the coating of wells with specific antigens or antibodies. Blocking agents were then used to prevent nonspecific binding, followed by incubation and washing steps. Enzyme-conjugated secondary antibodies were applied, and after further incubation and washing, a substrate solution was added to induce a colorimetric reaction. The optical density (OD) was measured using an ELISA plate reader to determine the concentration of the target proteins. Immunohistochemistry and histopathological examination Immunohistochemistry (IHC) was performed to analyze HIF-1α expression, involving fixation, antigen retrieval, blocking, and antibody incubation. Hematoxylin and eosin (H&E) staining was used to assess tissue structure and pathology. Samples were formalin-fixed, dehydrated, paraffin-embedded, sectioned with a microtome, and deparaffinized. After H&E staining, slides were hydrated, mounted, and examined under a light microscope. Images of the coaptation area were captured at 100× magnification, with six fields per slide (one central and five peripheral) to quantify axon density and capillary number. (27,33) Quantitative reverse transcriptase polymerase chain reaction (qRT-PCR) qRT-PCR was used to assess axonal density (NeuN), capillary formation (CD31), fibrosis (α-SMA), and glial markers (NCAM) in peripheral nerves, with α-actin as the housekeeping gene. Total RNA was extracted from sciatic nerve tissue using Tri RNA Reagent, treated with DNase I, and evaluated for purity (A260/A280 = 1.8–2.1) and integrity (RQI ≥ 7.5). cDNA was synthesized using the ExcelRT™ Kit, and qPCR was performed with the SensiFAST™ SYBR® Lo-ROX Kit on a Bio-Rad CFX system (95°C 2 min; 40 cycles of 95°C 10 s, 60°C 20 s). Melt curve analysis confirmed specificity, and reactions were run in technical duplicates to ensure reproducibility. Primer sequences were as follows: CD31 (F-CCCAGTGACATTCACAGACA and R-ACCTTGACCCTCAGGATCTC), NCAM (F-ACCATACTCCAGCACAGCACAG and R-AGCGACTTCCACTCAGCCTTG), α-SMA (F-GGCATCCACGAAACCACCTAT and R-CCTTCTGCATCCTGTCAGCAA), and NeuN (F-GCAGGATGAAGCAGCACAGAC and R-TGAACCGGAAGGGGATGTTG). Walking track analysis All groups were examined for motor function through a walking tract analysis every week, including pre-neurectomy and weeks one to six, which were analyzed using the sciatic function index (SFI) formula. (34–36) Rats traverse a 150 cm long, 13 cm wide, and 15 cm high acrylic track. Footprints were acquired using ink and white paper and subsequently captured through photography for Image J software analysis. The calculation of SFI was performed using the formula by Bain et al. in 1989. (33,37) SFI of 0 indicates normal, and -100 indicates total decline. Statistical analysis Statistical analyses were conducted using IBM SPSS Statistics (version 29.0; IBM Corp. Armonk, NY, USA). Descriptive analysis was used to assess data distribution expressed in mean ± standard deviation and presented in the form of tables and graphs. The Shapiro-Wilk test was evaluated data normality, while Levene’s test was used to assess the homogeneity of variances. Between-group differences were analyzed using Mann-Whitney U tests or independent samples t-tests, depending on data distribution. Paired comparisons between week three and week six were assessed using Wilcoxon signed-rank tests or paired t-tests. P -values of < 0.05 were considered statistically significant. Results Effect of hypoxia-conditioned allogeneic Schwann-like cells and secretome administration on HIF-1α expression Based on the results of IHC in Figure 1, it can be seen that the two intervention groups that were given hypoxia-conditioned allogeneic SLCs and secretome expressed HIF-1α more clearly in the cytoplasm of peripheral nerve tissue when compared to the control group. HIF-1a was expressed equally comparing between the two intervention groups. In addition, the expression also appeared clearer at week six when compared to week three in the control group and two intervention groups. Effect of hypoxia-conditioned allogeneic Schwann-like cells and secretome administration on TGF- b , VEGF, α-SMA, CD-31, NCAM, and Neu-N According to the previously described results (Table 1), there was a statistically significant difference in TGF-b (p=0.002), α-SMA (p=0.002), NCAM (p=0.000), and Neu-N (p=0.012) between the intervention and control groups in week three. Over the course of that week, it was shown that the two groups who received hypoxia conditioned allogeneic SLCs had higher levels of TGF-b, NCAM, and Neu-N and lower levels of α-SMA than the control group. TGF-b (p=0.000), α-SMA (p=0.003), CD-31 (p=0.009), NCAM (p=0.000), and Neu-N (p=0.000) all showed statistically significant differences at week six. Compared to the control group, the two intervention groups had reduced α-SMA and higher levels of TGF-b, CD-31, NCAM, and Neu-N. There was no significantly difference in VEGF at weeks three and six or CD-31 at week three. In the meantime, there was no significantly difference in CD-31 at week three and VEGF at weeks three and six. According to time, there were statistically significant changes in TGF-b (p=0.028), α-SMA (p=0.042), CD-31 (p=0.003), NCAM (p=0.016), and Neu-N (p=0.000) between weeks 3 and 6 in both intervention groups (Table 2). The levels of TGF-b, α-SMA, CD-31, NCAM, and Neu-N were higher in the two intervention groups at week 6 compared to week 3. The only variables in the control group that exhibited a significant difference were TGF-b (p=0.027) and VEGF (p=0.001). Compared to week three, the control group's TGF-b and VEGF levels were shown to be higher. Effect of hypoxia-conditioned allogeneic Schwann-like cells and secretome administration on axon and microvascular density The fascicles are closely packed with axons and the epineurium in the cross-section of the peripheral nerves (Figure 2). In addition, the fascicles were surrounded by a number of microvascular structures. Both the number of microvascular cells and the axon density appeared to be higher in the two intervention groups than in the control group. Nonetheless, the axon density and microvascular count were nearly identical between the two intervention groups. Effect of hypoxic conditioned allogeneic Schwann-like cells and secretome administration on motor function improvement Figure 3 showed the footprint analysis results from the walking track before and after the surgical procedure. Table 3 showed that there was no significant difference in SFI between the three groups prior to the intervention. Additionally, it was explained that from week two to week six, there was a statistically significant difference (p <0.050) between the two intervention groups and the control group, with the hypoxic conditioned allogeneic SLCs group and secretome group consistently having higher SFI values than the control group. Figure 4 also showed differences in the sciatic function index between the control group, secretome, and hypoxic conditioned allogeneic Schwann-like cells at pre-intervention and weeks one through six. The comparison of results is illustrated in the box plot shown in Figure 5. Discussion This study demonstrated that the two intervention groups given hypoxia-conditioned allogeneic SLCs and secretome expressed HIF-1α more clearly in the cytoplasm of peripheral nerve tissue than the control group. Administration of hypoxic-conditioned allogeneic SLCs accelerated peripheral nerve regeneration in acute peripheral nerve injury, as evidenced by increased TGF-b levels, HIF-1α and NCAM expression, axonal density of peripheral nerves through expression of Neu-N protein, and number of capillaries through expression of CD-31; decreased expression of α-SMA; and improved motor function. This finding has significant implications for developing novel therapeutic strategies for treating acute peripheral nerve injuries in humans. The increased expression of TGF-b, HIF-1α, NCAM, Neu-N protein, and CD-31 also the decreased expression of α-SMA suggest that specific cellular and molecular pathways are involved in promoting nerve regeneration. If similar results are confirmed in clinical trials, it may be possible to employ hypoxic-conditioned allogeneic SLCs or other methods that target the identified mechanisms to speed up nerve regeneration and accelerate functional recovery in patients with acute peripheral nerve injuries. This study's findings may inspire the development of novel therapeutic strategies for treating peripheral nerve injuries in humans. Additionally, the study shows that giving mice hypoxic-conditioned allogeneic SLCs accelerates their motor performance and encourages neuron regeneration. For individuals who commonly have motor impairments due to peripheral nerve injury, this result is particularly important. In order to help people with nerve injuries regain motor control and functionality, new therapeutic options may be available if the observed improvement in motor function translates to humans. Adipose tissue-derived mesenchymal stem cells (AdMSCs) are a multipotent source of stem cells with immunosuppressive properties and low immunogenicity, making them appropriate for regenerative therapy, graft tolerance, and autoimmune prevention. They are also easily harvested with little morbidity, which is an advantage over other mesenchymal stem cell sources.(38) Nevertheless, their ability to proliferate and differentiate into neurogenic lineages such as glial cells (SLCs) is inferior to that of stem cells derived from nervous tissue, so PRP is utilized to accelerate these capabilities in vitro. The therapeutic potential of PRP is attributed to its content of growth factors like TGF-b, PDGF, VEGF, and SDF-1α, which have been shown to promote neurogenic differentiation and cell proliferation. (28,39) Schwann-like cells (SLCs) resemble real Schwann cells after being incubated in an induction medium for several days. Most studies have demonstrated the augmentation effect after being implanted in silicon conduit on the peripheral nerve regeneration process. In particular, transplantation of MSCs has been demonstrated to lessen muscle atrophy, promote axon growth and myelination, and minimize inflammation. (40) Preconditioning MSCs under hypoxic conditions (1%–7% oxygen), which more closely resemble natural tissue environments than usual normoxic culture (21%), improves their survival through the activation of HIF-1α and an Akt-dependent pathway. This approach also accelerates the secretion of proangiogenic factors and increases the expression of SDF-1 and its receptors, CXCR4 and CXCR7, thereby improving MSCs engraftment in vivo. (41,42) Additionally, hypoxic preconditioning boosts the paracrine activity of MSCs and has been successfully applied in treating conditions like myocardial infarction and diabetes-related erectile dysfunction. (43,44) The secretome refers to the complex mixture of bioactive molecules secreted by cells into their extracellular milieu. In the context of Schwann-like cells (SLCs) derived from mesenchymal stem cells, especially under hypoxic conditions, the secretome plays a pivotal role in modulating the microenvironment of peripheral nerve injury and accelerating regeneration.(45) Under hypoxia (typically 1–5% O₂), SLCs exhibit a shift in paracrine signaling, resulting in upregulation of trophic and immunomodulatory factors. This hypoxia-induced adaptation mimics the physiological environment of injured tissues and primes the cells to secrete a richer, more regenerative profile of factors.(31,46) A multipurpose cytokine, TGF-b is crucial for neuroprotection and peripheral nerve repair. In order to promote nerve regeneration at lesion sites, TGF-b may trigger Schwann cell reprogramming, alter immune cells, increase neuronal growth potential, and control blood-nerve barrier permeability. (47) Additionally, it stimulates M2 macrophages, which work with Schwann cells to promote axonal regeneration. (48) TGF-b influences Schwann cell proliferation and differentiation, shifting them from a myelinating to an activated state. (49,50) TGF-b may also have extensive biological effects during nerve regeneration through interaction with transcriptional co-regulators and Smad-dependent and -independent signaling. (51,52) Subsequently, HIF-1α plays a central role in neuronal regeneration by regulating injury-induced genes and promoting axonal regrowth. It accelerates VEGF expression, which supports nerve repair and angiogenesis. Hypoxia-a crucial condition during peripheral nerve injury-increases HIF-1α and NGF expression, both of which synergize in regeneration. (53,54) A myofibroblast marker α-SMA, is expressed in pericytes and perineurial cells in uninjured nerves, but its role after injury remains unclear. (55) Injury activates fibrotic pathways involving Schwann cells, fibroblasts, and macrophages that clear myelin and may contribute to collagen deposition. (56,57) Excess collagen alters nerve architecture and may hinder regeneration. Various cells-including Schwann cells, fibroblasts, pericytes, and macrophages-synthesize collagen during this process. In addition, endoneurial fibroblasts are also derived from neural crest SCs as well as Schwann cells. (55) AdMSCs have the effect of downregulating the pro-fibrotic marker α-SMA gene expression and upregulating the anti-fibrotic fibroblast growth factor and pro-VEGF angiogenic gene. (58) AdMSCs appear to have differential activity by remodeling scar fibrotic matrix, changing the balance between ECM deposition and degradation for deterioration to occur, even though they come from the same mesodermal layer of myofibroblasts. (58,59) NCAM plays an important role during peripheral nerve regeneration. NCAM mediates interactions between axons, and together with cell adhesion molecule L1, it mediates between Schwann cells and axons. Following peripheral nerve injury, NCAM and N-cadherin are upregulated at the proximal ends of nerves. L1 and NCAM were found at the distal end of transected adult peripheral nerves. (12,60) Moreover, additional research indicates that NCAM/CD56 is crucial for a number of functions, such as adhesion molecules involved in peripheral nerve development and renewal, synaptic plasticity, cognitive function, and myelin formation and maintenance. Axons must be guided toward their target organs by interactions between Schwann cells and regenerated axons via adhesion molecules. Polysialic acid (PSA), an anionic glycan linked to NCAM and present on the surface of Schwann cells and neurons, is one of the important chemicals involved. (61) CD31, also known as Platelet Endothelial Cell Adhesion Molecule-1 (PECAM-1), is a 130-kDa cell surface marker from the immunoglobulin (Ig) superfamily, expressed in vascular endothelial cells and involved in leukocyte transmigration, anti-apoptotic signaling, and cell adhesion. (62) Its role in peripheral nerve regeneration is linked to angiogenesis, supported by CD31 immunoreactivity expression patterns, indicating a strong connection between nerve regeneration and vascularization. (63) NeuN as a marker for post-mitotic neurons, is commonly used to identify neurons after injury or damage. It serves as an indicator of neuronal presence and is considered a surrogate marker for peripheral nerve injury, with increased expression observed in small CGRP-positive DRG neurons during inflammation-suggesting its involvement in specific nociceptive neuron populations. (64) Limitation of study This study has several drawbacks, such as not examining other variables involved in the peripheral nerve regeneration process due to acute injury, such as collagen I and II. In addition, this study did not examine the results of peripheral nerve regeneration through histopathological and neurophysiological examinations. Research over a longer period is also needed to obtain a more complete knowledge of the increasing and decreasing levels or expression of several cytokines. Conclusions This study concluded that hypoxic-conditioned allogeneic SLCs accelerate peripheral nerve regeneration in acute nerve injury, evidenced by increased TGF-β, HIF-1α, NCAM, Neu-N, and CD-31 expression, reduced α-SMA expression, greater axonal density, and improved motor function. These findings highlight key molecular pathways involved in nerve repair and suggest potential for developing targeted therapies. If replicated clinically, hypoxic-conditioned SLCs could offer a novel approach to accelerate nerve regeneration and restore motor function in patients with peripheral nerve injuries. Declarations Author Contribution TS and DNU contributed to conceptualization and project administration and took the lead in funding acquisition and investigation.TS and R were responsible for data curation, formal analysis, validation, and visualization.R and HS contributed to methodology and resources.CRSP, DT, HBN, NA, FAR, SR, MFI, and FM contributed to investigation and resources.TS and R supervised the project.TS, R, and HS wrote the original draft of the manuscript.TS, R, DNU, DT, NA, FAR, SR, and MFI reviewed and edited the manuscript.“All authors read and approved the final manuscript.” Acknowledgement The authors thank drh Deya Karsari and drh Igo Syaiful Ihsan from the Stem Cell Research and Development Center, Airlangga University, Surabaya, Indonesia, Amed Gatut Guntoro for designing the graphical abstract, and Ms. Agisa Prawesti for assisting in arranging this study, providing technical/laboratory support, and animal care/testing support. 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Molecular and Cellular Neuroscience CD31 + cell transplantation promotes recovery from peripheral neuropathy. Molecular and Cellular Neuroscience [Internet]. 2014;62:60–7. Available from: http://dx.doi.org/10.1016/j.mcn.2014.08.005 Dömer P, Kayal J, Bienhold UJ, Kewitz B, Kretschmer T, Heinen C. Rapid and efficient immunomagnetic isolation of endothelial cells from human peripheral nerves. Scientific Reports [Internet]. 2021;11(1951):1–10. Available from: https://doi.org/10.1038/s41598-021-81361-x Anderson MB, Das S, Miller KE. Subcellular localization of neuronal nuclei (NeuN) antigen in size and calcitonin gene-related peptide (CGRP) populations of dorsal root ganglion (DRG) neurons during acute peripheral inflammation. Neurosci Lett. 2021;760:1–21. Tables Table 1 Differences in TGF-b, VEGF, α-SMA, CD-31, NCAM, and Neu-N values between the two intervention groups (hypoxic conditioned allogeneic Schwann-like cells and secretome) and the control group at weeks three and six. TGF- b VEGF α-SMA CD-31 NCAM Neu-N Week 3 Control 37,67±2,31 151,67±48,60 29,94±3,27 26,57±4,28 36,57±4,96 76,16±15,62 Intervention 1 113,60±14,03 203,00±112,46 18,70±2,56 25,01±2,44 63,01±3,46 104,30±7,25 Intervention 2 130,33±24,69 201,67±31,79 14,09±1,48 35,88±2,25 52,64±5,96 74,80±1,90 p-value 0.002d* 0.235d 0.000c* 0.003c* 0.000c* 0.049e* Week 6 Control 69,33±5,51 220,33±13,05 30,07±4,09 27,54±1,30 49,14±3,32 87,71±5,66 Intervention 1 172,80±11,76 239,00±41,89 22,15±1,87 34,64±3,66 77,20±5,39 166,41±10,85 Intervention 2 196,00±10,58 297,33±25,11 21,52±2,78 40,63±1,87 73,93±3,91 127,54±12,21 p-value 0.000c* 0.323c 0.003c* 0.000c* 0.000c* 0.000c* a Independent T test b Mann-Whithney c Anova test d Kruskal Wallis e Brown-Forsythe *p considered statistically significant Intervention 1 = SLCs Intervention 2 = secretome SLCs Table 2 Differences in TGF-b, VEGF, α-SMA, CD-31, NCAM, and Neu-N values between week three and six in the two intervention groups (hypoxic conditioned allogeneic Schwann-like cells and secretome) and the control group. TGF- b VEGF α-SMA CD-31 NCAM Neu-N Control Group Week 3 37,67±2,31 151,67±48,60 29,94±3,27 26,57±4,28 36,57±4,96 76,16±15,62 Week 6 69,33±5,51 220,33±13,05 30,07±4,09 27,54±1,30 49,14±3,32 87,71±5,66 p-value 0.002a* 0.043b* 0.966b 0.615b 0.010b* 0.168b Intervention 1 Group Week 3 113,60±14,03 203,00±112,46 18,70±2,56 25,01±2,44 63,01±3,46 104,30±7,25 Week 6 172,80±11,76 239,00±41,89 22,15±1,87 34,64±3,66 77,20±5,39 166,41±10,85 p-value 0.002a* 0.065a 0.030b* 0.001b* 0.001b* 0.000b* Intervention 2 Group Week 3 130,33±24,69 201,67±31,79 14,09±1,48 35,88±2,25 52,64±5,96 74,80±1,90 Week 6 196,00±10,58 297,33±25,11 21,52±2,78 40,63±1,87 73,93±3,91 127,54±12,21 p-value 0.001b* 0.008b* 0.015b* 0.048b* 0.007b* 0.002b* a Mann-Whithney and b Independent T-test were performed; *p considered statistically significant. Intervention 1 = SLCs Intervention 2 = secretome SLCs Table 3 Differences in sciatic function index between the two intervention groups (hypoxic conditioned allogeneic Schwann-like cells and secretome) and the control group at pre-intervention and weeks one to six. Pre Week 1 Week 2 Week 3 Week 4 Week 5 Week 6 Control -7,79±2,37 -73,27±7,76 -66,82±5,05 -60,49±7,86 -56,43±8,02 -54,99±7,53 -38,47±5,79 Intervention 1 -11,27±4,43 -70,4±6,57 -60,12±4,01 -49,37±7,12 -44,6±4,89 -37,83±4,49 -23,13±3,23 Intervention 2 -8,45±1,82 -64,52±6,2 -50,01±11,52 -31,91±7,11 -25,07±3,1 -19,74±0,69 -15,18±2,5 p-value 0.091a 0.341a 0.006b* 0.001a* 0.012a* 0.001a* 0.004b* a Independent T-test and b Mann-Whitney were performed; *p considered statistically significant. Intervention 1 = SLCs Intervention 2 = secretome SLCs Additional Declarations No competing interests reported. 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University","correspondingAuthor":false,"prefix":"","firstName":"Ferdiansyah","middleName":"","lastName":"Mahyudin","suffix":""}],"badges":[],"createdAt":"2025-12-22 09:54:10","currentVersionCode":1,"declarations":"","doi":"10.21203/rs.3.rs-8423634/v1","doiUrl":"https://doi.org/10.21203/rs.3.rs-8423634/v1","draftVersion":[],"editorialEvents":[],"editorialNote":"","failedWorkflow":false,"files":[{"id":102574552,"identity":"ad628b33-2423-4b1d-9a75-966c68e379d8","added_by":"auto","created_at":"2026-02-13 08:00:35","extension":"jpg","order_by":1,"title":"Figure 1","display":"","copyAsset":false,"role":"figure","size":2277812,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eExpression of HIF-1α on IHC.\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003e(A) Control group at week three, (B) Control group at week six, (C) Intervention group with hypoxia-conditioned allogeneic SLCs at week three, (D) Intervention group with hypoxia-conditioned allogeneic SLCs at week six. (E) Intervention group with hypoxia-conditioned allogeneic SLCs secretome at week three, and (F) Intervention group with hypoxia-conditioned allogeneic SLCs secretome at week six.\u003c/p\u003e\n\u003cp\u003eWhite arrows indicate the presence of microvascular, while yellow arrows indicate cytoplasm expressing HIF-1α.\u003c/p\u003e","description":"","filename":"Figure1.jpg","url":"https://assets-eu.researchsquare.com/files/rs-8423634/v1/80a62fea818cb8c26917542c.jpg"},{"id":102574553,"identity":"cf7141c5-8524-4e26-971c-c5b66d1b36d7","added_by":"auto","created_at":"2026-02-13 08:00:35","extension":"jpg","order_by":2,"title":"Figure 2","display":"","copyAsset":false,"role":"figure","size":1386110,"visible":true,"origin":"","legend":"\u003cp\u003eBox plots showing the average serum levels of TGF-β and VEGF, as well as the average expression levels of α-SMA, NCAM, NeuN, and CD31 in each group.\u003c/p\u003e","description":"","filename":"Figure2.jpg","url":"https://assets-eu.researchsquare.com/files/rs-8423634/v1/f1c96fe9df17ca6a9acd7dd0.jpg"},{"id":102574557,"identity":"59d867e7-e7b3-478f-be0d-5ba6c2f797fb","added_by":"auto","created_at":"2026-02-13 08:00:35","extension":"jpg","order_by":3,"title":"Figure 3","display":"","copyAsset":false,"role":"figure","size":5973432,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eHistopathological cross-sectional images of the distal sciatic nerve with 100 times magnification.\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003e(A) Control group at week three, (B) Control group at week six, (C) Intervention group with hypoxia-conditioned allogeneic SLCs at week three, (D) Intervention group with hypoxia-conditioned allogeneic SLCs at week six, (E) Intervention group with hypoxia-conditioned allogeneic SLCs secretome at week three, and (F) Intervention group with hypoxia-conditioned allogeneic SLCs secretome at week six.\u003c/p\u003e\n\u003cp\u003eYellow arrows indicate the presence of microvascular, blue arrows indicate axons, and black arrows indicate epineurium.\u003c/p\u003e","description":"","filename":"Figure3.jpg","url":"https://assets-eu.researchsquare.com/files/rs-8423634/v1/12e0692712ade7596e9d6513.jpg"},{"id":102574554,"identity":"6449ec4c-67a4-4bab-aff0-52dd48ceb779","added_by":"auto","created_at":"2026-02-13 08:00:35","extension":"jpg","order_by":4,"title":"Figure 4","display":"","copyAsset":false,"role":"figure","size":297184,"visible":true,"origin":"","legend":"\u003cp\u003eThe results of the footprints from the walking track analysis are (A) before and (B) after the surgical treatment.\u003c/p\u003e","description":"","filename":"Figure4.jpg","url":"https://assets-eu.researchsquare.com/files/rs-8423634/v1/ae8dfa594782edf8074f5afa.jpg"},{"id":102747339,"identity":"bc46ab07-da02-4e90-a110-62dd1a0c0022","added_by":"auto","created_at":"2026-02-16 09:04:33","extension":"jpg","order_by":5,"title":"Figure 5","display":"","copyAsset":false,"role":"figure","size":109373,"visible":true,"origin":"","legend":"\u003cp\u003eDifferences in sciatic functional index (SFI) between the two intervention groups (hypoxic conditioned allogeneic Schwann-like cells and secretome), and control group at pre-intervention and weeks one to six.\u003c/p\u003e","description":"","filename":"Figure5.jpg","url":"https://assets-eu.researchsquare.com/files/rs-8423634/v1/48d4102ea56329f49500a154.jpg"},{"id":102962208,"identity":"31e42495-e23d-49ae-9e21-99c2e60f282a","added_by":"auto","created_at":"2026-02-19 04:05:29","extension":"pdf","order_by":0,"title":"","display":"","copyAsset":false,"role":"manuscript-pdf","size":11353252,"visible":true,"origin":"","legend":"","description":"","filename":"manuscript.pdf","url":"https://assets-eu.researchsquare.com/files/rs-8423634/v1/6c1e6fec-b1e9-412c-a00a-c3d0bb3c711d.pdf"},{"id":102747215,"identity":"52167d37-732f-4fa4-9cf9-342c6085bdbe","added_by":"auto","created_at":"2026-02-16 09:04:12","extension":"docx","order_by":1,"title":"","display":"","copyAsset":false,"role":"supplement","size":309478,"visible":true,"origin":"","legend":"","description":"","filename":"GraphicalAbstract.docx","url":"https://assets-eu.researchsquare.com/files/rs-8423634/v1/5cbe088cda6df9378cc2a983.docx"}],"financialInterests":"No competing interests reported.","formattedTitle":"Augmentation of peripheral nerve regeneration by hypoxic allogenic Schwann-like cells in acute nerve injury of Rattus norvegicus","fulltext":[{"header":"Significance Statement","content":"\u003cp\u003ePeripheral nerve injuries often cause lasting disability due to poor regeneration and surgical results. This study shows that transplanting hypoxic-conditioned allogeneic Schwann-like cells (SLCs) significantly enhances nerve repair in acute injuries. Hypoxic preconditioning increased key regenerative factors\u0026mdash;HIF-1\u0026alpha;, TGF-\u0026beta;, NCAM, Neu-N, and CD-31\u0026mdash;and decreased \u0026alpha;-SMA, leading to higher axon density, better blood vessel growth, and improved motor recovery. These findings indicate that hypoxic-conditioned SLCs and their secretions are promising, scalable options for faster nerve healing, potentially serving as effective adjuncts to microsurgery and improving patient outcomes.\u003c/p\u003e"},{"header":"Introduction","content":"\u003cp\u003ePeripheral nerve injury (PNI) may cause permanent sensory dysfunction and severe motor impairment. The consequences of these injuries are often severe, affecting daily living and even devastating.(1)\u003csup\u003e\u0026nbsp;\u003c/sup\u003eAnnually, PNI caused by accidents and trauma is 200,000 in the United States and 300,000 in Europe.(2,3)\u0026nbsp;A retrospective study in Surabaya, Indonesia, also showed that brachial plexus injuries were prevalent, especially in men and the 21-30 age group.(4)\u0026nbsp;Another study conducted in Surakarta, Indonesia, also provides a similar description, with the most common etiology being traffic accidents, especially motorcycle-related ones.(5)\u0026nbsp;\u003c/p\u003e\n\u003cp\u003eTraumatic peripheral nerve injury (PNI) remains difficult to manage despite its high prevalence. The current gold standard-nerve grafting with epineural microsuturing\u0026mdash;requires advanced skill yet often yields suboptimal outcomes due to inflammation, scar formation, axonal misdirection, and slow regeneration.(6\u0026ndash;8)\u003c/p\u003e\n\u003cp\u003eNew strategies are needed to accelerate axonal regeneration and functional recovery. Cell-based augmentation therapy offers promise by creating a supportive microenvironment forrepair.(9,10) Among candidate cells, mesenchymal stem cells (MSCs)-notably bone marrow- (BM-MSCs) and adipose-derived (AdMSCs)-stand out for their self-renewal, multipotency, and paracrine effects.(11,12) Schwann cells remain the natural choice for nerve repair but are limited by donor-site morbidity, slow growth, and difficulty in culture.(13,14)\u003c/p\u003e\n\u003cp\u003eAdMSCs can be induced into Schwann-like cells (SLCs), expressing Schwann cell markers and promoting neurite extension, myelination, and neuronal survival via neurotrophic factor secretion and recruitment of endogenous Schwann cells.(15,16)\u003c/p\u003e\n\u003cp\u003eHypoxic preconditioning (HPC), which cultures cells under 1\u0026ndash;5% O₂ before transplantation, mimics the injured tissue milieu and accelerates survival, angiogenesis, and neuroprotection through HIF-1\u0026alpha;\u0026ndash;mediated pathways, generating a more robust regenerative phenotype.(17,18)\u003c/p\u003e\n\u003cp\u003eThe secretome-the spectrum of bioactive molecules released by hypoxia-conditioned SLCs-plays a pivotal role in modulating the injury microenvironment, accelerating axonal regrowth, and supporting functional recovery after PNI.(19,20)\u003c/p\u003e\n\u003cp\u003eThis study aims to investigate the augmentation of nerve regeneration after administration of hypoxic conditioned allogeneic SLCs and its secretome in peripheral nerve reinnervation by analyzing several markers, including the expression of HIF-1\u0026alpha;, CD-31, and Neu-N protein, \u0026alpha;-SMA, and NCAM; TGF-b and VEGF levels; and improvement of motor function with walking track analysis.\u003c/p\u003e"},{"header":"Materials \u0026 Methods","content":"\u003cp\u003e\u003cstrong\u003eStudy design\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThis in-vivo experimental study uses white rats (\u003cem\u003e\u003cu\u003eRattus\u003c/u\u003e\u003c/em\u003e \u003cem\u003e\u003cu\u003enorvegicus\u003c/u\u003e\u003c/em\u003e) Wistar strain, obtained from the Animal and Stem Cell Research Institute of the Tropical Disease Laboratory, Airlangga University, Surabaya, East Java, Indonesia.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003eIsolation and culture of AdMSCs, preparation of hypoxic SLCs, surgery process (including denervation and reinnervation), and administration of hypoxic SLCs were also carried out there. The process of tissue preparations, immunohistochemical staining, observing micrografts, collecting data, and validating observational data was carried out at the Animal and Stem Cell Research Institute of Tropical Disease Laboratory, Airlangga University, Surabaya, East Java, Indonesia, as well as the Anatomy-Histology Laboratory, and the Anatomical Pathology Laboratory, Faculty of Medicine, Public Health, and Nursing, Gadjah Mada University, Yogyakarta, Indonesia, conducted from May to July 2022.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003eThis study was conducted in accordance with the Animal in Research: Reporting In Vivo Experiments (ARRIVE) 2.0 guidelines, and a completed checklist has been submitted as supplementary material.(21)\u003c/p\u003e\n\u003cp\u003eThe ethical assessment of this research was considered by the Animal Care and Use Committee (ACUC), Faculty of Veterinary Medicine, Airlangga University, Surabaya, East Java, Indonesia, and received an ethical clearance with a letter-number of 2.KE.038.05.2020 on May 13, 2020.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAnimal\u003c/strong\u003e \u003cstrong\u003emodel\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThis study use the white male rat\u0026nbsp;\u003cem\u003e\u003cu\u003eRattus\u003c/u\u003e\u003c/em\u003e \u003cem\u003e\u003cu\u003enorvegicus\u003c/u\u003e\u003c/em\u003e Wistar strain with aged 3-4 months, weighing 200-300 grams (to make the sample more homogeneous), and healthy. The health status was analyzed by examination to active movements, no skin wounds, no red eyes, respiratory rate \u0026lt;60 times per minute, and rectal temperature \u0026lt; 40\u0026deg;C, which was confirmed by a competent veterinarian. The number of replicated experimental is rounded 6 units, determined the sample size formula by Lemeschow\u003cspan lang=\"EN-US\"\u003e(22)\u003c/span\u003e, with calculating correction factor. \u0026nbsp;\u003c/p\u003e\n\u003cp\u003eThe drop-out criteria in this study were animals that died during the research process.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003eSubsequently, this study divided the research subjects into three groups, one group is control and two groups were intervention group. The control group received denervation and reinnervation with sutures. The intervention group received the denervation and reinnervation group with sutures plus allogeneic SLCs under hypoxic conditions, and their secretome. Animals were randomly allocated to control and intervention groups using a computer-generated random sequence to minimise allocation bias. Blinding was applied throughout the study: the personnel conducting the experiment, outcome assessment, and data analysis were unaware of group allocations.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAnimal\u003c/strong\u003e \u003cstrong\u003epreparation\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eTwenty-four \u003cem\u003e\u003cu\u003eRattus\u003c/u\u003e\u003c/em\u003e \u003cem\u003e\u003cu\u003enorvegicus\u003c/u\u003e\u003c/em\u003e were placed in standard cages (measuring 40x60x15cm) made of plastic trays covered with husks and woven wire and had partitions so that the animals could not mix. The housing density was maintained at two rats per cage to ensure sufficient space and reduce competition for resources. Environmental enrichment included nesting materials and chew toys to promote natural behaviors and improve animal welfare. The cage is hygienic and shielded from wind, rain, and direct sunlight while maintaining an ambient temperature of approximately 15-20\u0026deg;C. Furthermore, the cages were given sufficient lighting with light-dark cycles per 12 hours at 24\u0026deg;C and 50% humidity and maintained under standard laboratory conditions with routine veterinary oversight. Housing was designed to provide both physical and psychological comfort, with regular cleaning schedules and monitoring to prevent overcrowding and ensure a stress-free environment. Experimental animals were also given food and drink \u003cem\u003ead\u003c/em\u003e \u003cem\u003elibitum\u003c/em\u003e. The rat meal extract contains 20-25% protein, 5% fat, 40-50% starch, and 5% crude fiber. Each day, 12-20 grams of food and 80-100 cc/kg water was administered. No animals/data were excluded from the analysis.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAnesthetic, euthanasia, and post-experimental procedures\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eAll invasive procedures, including surgeries and sample collections, were performed under general anesthesia to minimize pain and distress. Rats were anesthetized using an intramuscular injection of 50-55 mg/kg Ketamine hydrochloride (@Ket-A-100) and 25 mg/kg Xylazine hydrochloride (@Xyla 20mg). The depth of anesthesia was confirmed by the absence of reflex responses to stimuli. Post-operative analgesia was included in the experimental protocol to minimize pain and support animal welfare, where feasible. Following surgery, animals received intramuscular injections of Phenylbutazone at a dose of 20 mg/kg once daily for up to three days, based on clinical assessment and antibiotics Enrofloxacin 20-40mg/kg before surgey and for 2 days after surgery as prophylaxis. Animals were monitored daily for signs of pain or distress, and additional analgesic doses were administered if necessary. Subsequently, animals used for blood and tissue sampling were euthanized after the completion of sample collection to prevent post-surgical discomfort. Last, After completion of the experimental procedures, all surviving rats were humanely euthanized with a lethal dose of sodium pentobarbital administered via the carotid artery under deep anesthesia. This method was selected to ensure a rapid and painless death in accordance with AVMA guidelines on euthanasia. This also prevents prolonged captivity and ensures no unnecessary suffering. No animals remained alive post-experiment, adhering to ethical guidelines for the humane treatment of laboratory animals.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003ePreparation of stromal vascular fraction, isolation and culture of AdMSCs\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eTwo experimental animals \u003cem\u003e\u003cu\u003eRattus\u003c/u\u003e\u003c/em\u003e \u003cem\u003e\u003cu\u003enorvegicus\u003c/u\u003e\u003c/em\u003e were used to isolate AdMSCs. Animals were kept in a controlled environment. The rats were euthanized, and the abdominal fat pad was extracted. Adipose-derived mesenchymal stem cells (AdMSCs) were isolated using the methodology described by Zuk et al. (2001) and Gimble et al. (2007).(23,24)\u0026nbsp;\u003c/p\u003e\n\u003cp\u003eSubsequently, to verify the cellular identity of cultivated AdMSCs cells, immunofluorescence labeling was performed on AdMSCs from passage 3 using CD14, CD29, CD34, CD44, CD45, CD73, CD90, and CD105 markers.\u003cspan lang=\"EN-US\"\u003e(25,26)\u003c/span\u003e\u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003ePreparation of platelet-rich plasma\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eBlood was collected from three anesthetized rats via the carotid artery. Following blood collection, the animals were euthanized. 40 mL of blood was immediately mixed with citrate phosphate dextrose (CPD) buffer as an anticoagulant, using a 1:5 ratio (1 mL CPD to 5 mL blood), with a CPD concentration of 0.15 mg/mL. The blood sample was centrifuged at a force of 500 g for 5 minutes using the Heraeus Cryfuge 6000i centrifuge from Kendro Laboratory Products in Hessen, Germany. Subsequently, the liquid portion with a high concentration of platelets, known as platelet-rich plasma (PRP), was subjected to a second round of centrifugation at a force of 2000 times the acceleration due to gravity (2000g) for 5 minutes. Subsequently, the platelet pellets from the second spin were resuspended with the supernatant until a final concentration of 1.5 \u0026times; 10\u0026sup1;\u0026sup2; platelets/L was achieved.(27)\u003csup\u003e\u0026nbsp;\u0026nbsp;\u003c/sup\u003ePRP is needed for the preparation of Schwann-like cells.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003ePreparation of Schwann-like cells (neuronal/ glial trans-differentiation)\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe Kingham procedure was used to prepare SLCs produced from AdMSC rats, and a 10% PRP solution was administered.\u003cspan lang=\"EN-US\"\u003e(28)\u003c/span\u003e During the third pass, the AdMSCs cells were transformed into a phenotype similar to SLC-dAdMSCs through two stages. Initially, the culture medium was replaced with a medium containing 1 mM\u0026sup3; - mercaptoethanol (Scharlau Chemicals) for 24 hours. This was followed by treatment with 35 ng/mL all-trans retinoic acid (Sigma-Aldrich) for 72 hours. Subsequently, the cells were exposed to a differentiation medium containing 5 ng/mL PDGF, 10 ng/mL bFGF (both from PeproTech), 14 \u0026micro;M forskolin (Sigma-Aldrich), 252 ng/mL neuregulin-1 (R\u0026amp;D Systems), and 10% PRP. This differentiation was maintained for at least 14 days before the cells were characterized.\u003cspan lang=\"EN-US\"\u003e(28,29)\u003c/span\u003e Cells used in this study were from passages 3 to 6. Oxygen levels during culture were maintained at 21% using the ProOx-C-chamber 110 (Biospherix, Redfield, NY) under tightly controlled conditions for 24 hours.\u003cspan lang=\"EN-US\"\u003e(30)\u003c/span\u003e\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eIsolation of rat sciatic nerve-derived stem cells\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eSegments of sciatic nerve approximately 1 cm were dissected and cleared of the epineurium. The tissue was then cut into ~1 mm pieces and enzymatically digested using 2 mg/mL collagenase NB4. Then, dispersion was carried out in DMEM/F-12 for 15 minutes at 37\u0026deg;C, followed by centrifugation and resuspension in DMEM/F-12, which contained 10% FBS. Subsequently, the cells obtained were cultured at 37\u0026deg;C with 5% CO\u003csub\u003e2\u003c/sub\u003e atmosphere. After 48 hours, the supernatant was discarded, and the remaining cells were treated with 1 mg/mL collagenase NB4 to separate them from the fibroblast cells.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eHypoxic pre-condition treatment on Schwann-like cells\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eHypoxic preconditioning treatment was performed on SLCs and rat sciatic nerve-derived SCs with O\u003csub\u003e2\u003c/sub\u003e levels of 1%, 3%, and 5% in preliminary studies.\u003cspan lang=\"EN-US\"\u003e(31)\u003c/span\u003e Hypoxic preconditioning is carried out by stimulating in a water-saturated gas mixture at 1%, 3% or 5% of O\u003csub\u003e2\u003c/sub\u003e, 5% of CO\u003csub\u003e2\u003c/sub\u003e, and 94% N\u003csub\u003e2\u003c/sub\u003e at 37\u0026deg;C for 24 hours. The levels of several growth factors were examined, including BDNF, GDNF, bFGF, NGF, TGF-b, and VEGF, using the ELISA from SLCs and SCs, respectively, in 21% normoxia and 1%, 3%, and 5% hypoxic conditions, which result that no significant differences in growth factor secretion were observed between SLCs and SCs. Furthermore, growth factor concentrations were observed in SLCs for all oxygen concentrations, and the result was that 1% oxygen content had the highest growth factor concentration. This research has been carried out in previous studies,\u003cspan lang=\"EN-US\"\u003e(31)\u003c/span\u003e so that SLCs with 1% hypoxic conditions were used for this study. The 1% hypoxic preconditioning of SLC-dAdMSCs was performed in a controlled ProOx-C-chamber system (ProOx 110; Biospherix, Redfield, NY) for 24 hours.\u003cspan lang=\"EN-US\"\u003e(30)\u003c/span\u003e\u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eSecretome production from cell cultures under h\u003c/strong\u003e\u003cstrong\u003eypoxic\u003c/strong\u003e\u003cstrong\u003e\u0026nbsp;conditions\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003ePassage/sub-culture of cells that have reached 70-80% confluence for propagation. Re-seeding or re-planting the cell culture on a 100 mm petri dish with a density of 5x10\u003csup\u003e5\u003c/sup\u003e cells per 100 mm petri dish. Incubate the grown cells in a 1% hypoxic incubator for 4 days. Collect the supernatant/medium from the petri dish and collect it in a connical tube. Centrifuge at 3000 rpm for 5 minutes. Filter the supernatant using a 0.45 \u0026micro;m pore filter and collect it in a new connical tube. Store the supernatant (secretome) at -20\u003csup\u003eo\u003c/sup\u003eC\u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eDenervation and reinnervation procedure\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eAll surgical procedures were performed under anesthesia to ensure humane treatment of the animals. In the control group, the sciatic nerve was denervated by cutting the sciatic nerve (neurectomy), which was opened through the gluteal region of the leg and then reinnervated with sutures using 10-0 nylon thread after waiting for 10 minutes by giving the amniotic membrane to the nerve reinnervation area. In the two intervention groups, the sciatic nerve was denervated, then reinnervated with sutures using 10-0 nylon thread and given allogeneic SLCs under 1% hypoxic conditions, and one group another with secretome of allogeneic SLCs under 1% hypoxic conditions. The amnion membrane was used as a scaffold in the nerve reinnervation area. The technique of joining the proximal to the distal nerve section was performed using microsurgery with four interrupted sutures on the epineural using 10-0 polypropylene monofilament.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAnimal dissection and nerve extraction\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eExperimental animals were anesthetized using intramuscular injection of 50-55 mg/kg Ketamine hydrochloride (@Ket-A-100) and 25mg/kg xylazine hydrochloride (@Xyla 20mg). After skin disinfection, the hair was scraped off with a 10% povidone-iodine solution. The right sciatic nerve was accessed via a skin incision and carefully isolated using a gluteal muscle-splitting approach. The left leg served as an internal control. The sciatic nerve axonotomesis procedure in the mid-hind legs of rats by Savastano (2014) was used in this study.\u003cspan lang=\"EN-US\"\u003e(32)\u003c/span\u003e All surgery processes were performed by the same investigator, using microsurgery loops and tools.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eEnzyme-linked immunosorbent assay (ELISA) examination\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eTo assess the serum levels of TGF-b and VEGF, enzyme-linked immunosorbent assay (ELISA) was performed. The procedure began with the coating of wells with specific antigens or antibodies. Blocking agents were then used to prevent nonspecific binding, followed by incubation and washing steps. Enzyme-conjugated secondary antibodies were applied, and after further incubation and washing, a substrate solution was added to induce a colorimetric reaction. The optical density (OD) was measured using an ELISA plate reader to determine the concentration of the target proteins.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eImmunohistochemistry and histopathological examination\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eImmunohistochemistry (IHC) was performed to analyze HIF-1\u0026alpha; expression, involving fixation, antigen retrieval, blocking, and antibody incubation. Hematoxylin and eosin (H\u0026amp;E) staining was used to assess tissue structure and pathology. Samples were formalin-fixed, dehydrated, paraffin-embedded, sectioned with a microtome, and deparaffinized. After H\u0026amp;E staining, slides were hydrated, mounted, and examined under a light microscope. Images of the coaptation area were captured at 100\u0026times; magnification, with six fields per slide (one central and five peripheral) to quantify axon density and capillary number.\u003cspan lang=\"EN-US\"\u003e(27,33)\u003c/span\u003e\u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eQuantitative reverse transcriptase polymerase chain reaction (qRT-PCR)\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eqRT-PCR was used to assess axonal density (NeuN), capillary formation (CD31), fibrosis (\u0026alpha;-SMA), and glial markers (NCAM) in peripheral nerves, with \u0026alpha;-actin as the housekeeping gene. Total RNA was extracted from sciatic nerve tissue using Tri RNA Reagent, treated with DNase I, and evaluated for purity (A260/A280 = 1.8\u0026ndash;2.1) and integrity (RQI \u0026ge; 7.5). cDNA was synthesized using the ExcelRT\u0026trade; Kit, and qPCR was performed with the SensiFAST\u0026trade; SYBR\u0026reg; Lo-ROX Kit on a Bio-Rad CFX system (95\u0026deg;C 2 min; 40 cycles of 95\u0026deg;C 10 s, 60\u0026deg;C 20 s). Melt curve analysis confirmed specificity, and reactions were run in technical duplicates to ensure reproducibility. Primer sequences were as follows:\u0026nbsp;\u003c/p\u003e\n\u003cp\u003eCD31 (F-CCCAGTGACATTCACAGACA and R-ACCTTGACCCTCAGGATCTC),\u0026nbsp;\u003c/p\u003e\n\u003cp\u003eNCAM (F-ACCATACTCCAGCACAGCACAG and R-AGCGACTTCCACTCAGCCTTG), \u0026alpha;-SMA (F-GGCATCCACGAAACCACCTAT and R-CCTTCTGCATCCTGTCAGCAA), and NeuN (F-GCAGGATGAAGCAGCACAGAC and R-TGAACCGGAAGGGGATGTTG).\u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eWalking track analysis\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eAll groups were examined for motor function through a walking tract analysis every week, including pre-neurectomy and weeks one to six, which were analyzed using the sciatic function index (SFI) formula.\u003cspan lang=\"EN-US\"\u003e(34\u0026ndash;36)\u003c/span\u003e Rats traverse a 150 cm long, 13 cm wide, and 15 cm high acrylic track. Footprints were acquired using ink and white paper and subsequently captured through photography for Image J software analysis. The calculation of SFI was performed using the formula by Bain et al. in 1989.\u003cspan lang=\"EN-US\"\u003e(33,37)\u003c/span\u003e SFI of 0 indicates normal, and -100 indicates total decline.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eStatistical analysis\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eStatistical analyses were conducted using IBM SPSS Statistics (version 29.0; IBM Corp. Armonk, NY, USA). Descriptive analysis was used to assess data distribution expressed in mean \u0026plusmn; standard deviation and presented in the form of tables and graphs. The Shapiro-Wilk test was evaluated data normality, while Levene\u0026rsquo;s test was used to assess the homogeneity of variances. Between-group differences were analyzed using Mann-Whitney U tests or independent samples t-tests, depending on data distribution. Paired comparisons between week three and week six were assessed using Wilcoxon signed-rank tests or paired t-tests. \u003cem\u003eP\u003c/em\u003e-values of \u0026lt; 0.05 were considered statistically significant.\u003c/p\u003e"},{"header":"Results","content":"\u003cp\u003e\u003cstrong\u003eEffect of hypoxia-conditioned allogeneic Schwann-like cells and secretome administration on\u0026nbsp;\u003c/strong\u003e\u003cstrong\u003eHIF-1\u0026alpha;\u003c/strong\u003e\u003cstrong\u003e\u0026nbsp;\u003c/strong\u003e\u003cstrong\u003eexpression\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eBased on the results of IHC in Figure 1, it can be seen that the two intervention groups that were given hypoxia-conditioned allogeneic SLCs and secretome expressed HIF-1\u0026alpha; more clearly in the cytoplasm of peripheral nerve tissue when compared to the control group. HIF-1a was expressed equally comparing between the two intervention groups.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003eIn addition, the expression also appeared clearer at week six when compared to week three in the control group and two intervention groups.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eEffect\u003c/strong\u003e \u003cstrong\u003eof\u003c/strong\u003e \u003cstrong\u003ehypoxia-conditioned\u003c/strong\u003e \u003cstrong\u003eallogeneic\u003c/strong\u003e \u003cstrong\u003eSchwann-like\u003c/strong\u003e \u003cstrong\u003ecells\u003c/strong\u003e\u003cstrong\u003e\u0026nbsp;and\u0026nbsp;\u003c/strong\u003e\u003cstrong\u003esecretome\u003cem\u003e\u0026nbsp;\u003c/em\u003e\u003c/strong\u003e\u003cstrong\u003eadministration\u003c/strong\u003e \u003cstrong\u003eon\u003c/strong\u003e \u003cstrong\u003eTGF-\u003c/strong\u003e\u003cstrong\u003eb\u003c/strong\u003e\u003cstrong\u003e,\u0026nbsp;\u003c/strong\u003e\u003cstrong\u003eVEGF,\u003c/strong\u003e \u003cstrong\u003e\u0026alpha;-SMA, CD-31,\u003c/strong\u003e \u003cstrong\u003eNCAM,\u003c/strong\u003e \u003cstrong\u003eand\u003c/strong\u003e \u003cstrong\u003eNeu-N\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eAccording to the previously described results (Table 1), there was a statistically significant difference in TGF-b\u0026nbsp;(p=0.002), \u0026alpha;-SMA (p=0.002), NCAM (p=0.000), and Neu-N (p=0.012) between the intervention and control groups in week three. Over the course of that week, it was shown that the two groups who received hypoxia conditioned allogeneic SLCs had higher levels of TGF-b, NCAM, and Neu-N and lower levels of \u0026alpha;-SMA than the control group. TGF-b\u0026nbsp;(p=0.000), \u0026alpha;-SMA (p=0.003), CD-31 (p=0.009), NCAM (p=0.000), and Neu-N (p=0.000) all showed statistically significant differences at week six. Compared to the control group, the two intervention groups had reduced \u0026alpha;-SMA and higher levels of TGF-b, CD-31, NCAM, and Neu-N.\u003c/p\u003e\n\u003cp\u003eThere was no significantly difference in VEGF at weeks three and six or CD-31 at week three. In the meantime, there was no significantly difference in CD-31 at week three and VEGF at weeks three and six. According to time, there were statistically significant changes in TGF-b (p=0.028), \u0026alpha;-SMA (p=0.042), CD-31 (p=0.003), NCAM (p=0.016), and Neu-N (p=0.000) between weeks 3 and 6 in both intervention groups (Table 2). The levels of TGF-b, \u0026alpha;-SMA, CD-31, NCAM, and Neu-N were higher in the two intervention groups at week 6 compared to week 3. The only variables in the control group that exhibited a significant difference were TGF-b (p=0.027) and VEGF (p=0.001). Compared to week three, the control group\u0026apos;s TGF-b and VEGF levels were shown to be higher.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eEffect\u003c/strong\u003e \u003cstrong\u003eof\u003c/strong\u003e \u003cstrong\u003ehypoxia-conditioned\u003c/strong\u003e \u003cstrong\u003eallogeneic\u003c/strong\u003e \u003cstrong\u003eSchwann-like\u003c/strong\u003e \u003cstrong\u003ecells\u003c/strong\u003e \u003cstrong\u003eand\u0026nbsp;\u003c/strong\u003e\u003cstrong\u003esecretome\u003c/strong\u003e\u003cstrong\u003e\u0026nbsp;administration\u003c/strong\u003e \u003cstrong\u003eon\u003c/strong\u003e \u003cstrong\u003eaxon\u003c/strong\u003e \u003cstrong\u003eand\u0026nbsp;\u003c/strong\u003e\u003cstrong\u003emicrovascular\u003c/strong\u003e \u003cstrong\u003edensity\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe fascicles are closely packed with axons and the epineurium in the cross-section of the peripheral nerves (Figure 2). In addition, the fascicles were surrounded by a number of microvascular structures. Both the number of microvascular cells and the axon density appeared to be higher in the two intervention groups than in the control group. Nonetheless, the axon density and microvascular count were nearly identical between the two intervention groups.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eEffect\u003c/strong\u003e \u003cstrong\u003eof\u003c/strong\u003e \u003cstrong\u003ehypoxic\u003c/strong\u003e \u003cstrong\u003econditioned\u003c/strong\u003e \u003cstrong\u003eallogeneic\u003c/strong\u003e \u003cstrong\u003eSchwann-like\u003c/strong\u003e \u003cstrong\u003ecells\u003c/strong\u003e \u003cstrong\u003eand\u0026nbsp;\u003c/strong\u003e\u003cstrong\u003esecretome\u003c/strong\u003e\u003cstrong\u003e\u0026nbsp;administration\u003c/strong\u003e \u003cstrong\u003eon\u003c/strong\u003e \u003cstrong\u003emotor\u0026nbsp;\u003c/strong\u003e\u003cstrong\u003efunction\u003c/strong\u003e \u003cstrong\u003eimprovement\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eFigure 3 showed the footprint analysis results from the walking track before and after the surgical procedure. Table 3 showed that there was no significant difference in SFI between the three groups prior to the intervention. Additionally, it was explained that from week two to week six, there was a statistically significant difference (p \u0026lt;0.050) between the two intervention groups and the control group, with the hypoxic conditioned allogeneic SLCs group and secretome group consistently having higher SFI values than the control group. Figure 4 also showed differences in the sciatic function index between the control group, secretome, and hypoxic conditioned allogeneic Schwann-like cells at pre-intervention and weeks one through six. The comparison of results is illustrated in the box plot shown in Figure 5.\u003c/p\u003e"},{"header":"Discussion","content":"\u003cp\u003eThis study demonstrated that the two intervention groups given hypoxia-conditioned allogeneic SLCs and secretome expressed HIF-1\u0026alpha; more clearly in the cytoplasm of peripheral nerve tissue than the control group. Administration of hypoxic-conditioned allogeneic SLCs accelerated peripheral nerve regeneration in acute peripheral nerve injury, as evidenced by increased TGF-b\u0026nbsp;levels, HIF-1\u0026alpha; and NCAM expression, axonal density of peripheral nerves through expression of Neu-N protein, and number of capillaries through expression of CD-31; decreased expression of \u0026alpha;-SMA; and improved motor function. This finding has significant implications for developing novel therapeutic strategies for treating acute peripheral nerve injuries in humans. The increased expression of TGF-b, HIF-1\u0026alpha;, NCAM, Neu-N protein, and CD-31 also the decreased expression of \u0026alpha;-SMA suggest that specific cellular and molecular pathways are involved in promoting nerve regeneration.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003eIf similar results are confirmed in clinical trials, it may be possible to employ hypoxic-conditioned allogeneic SLCs or other methods that target the identified mechanisms to speed up nerve regeneration and accelerate functional recovery in patients with acute peripheral nerve injuries. This study\u0026apos;s findings may inspire the development of novel therapeutic strategies for treating peripheral nerve injuries in humans. Additionally, the study shows that giving mice hypoxic-conditioned allogeneic SLCs accelerates their motor performance and encourages neuron regeneration. For individuals who commonly have motor impairments due to peripheral nerve injury, this result is particularly important. In order to help people with nerve injuries regain motor control and functionality, new therapeutic options may be available if the observed improvement in motor function translates to humans.\u003c/p\u003e\n\u003cp\u003eAdipose tissue-derived mesenchymal stem cells (AdMSCs) are a multipotent source of stem cells with immunosuppressive properties and low immunogenicity, making them appropriate for regenerative therapy, graft tolerance, and autoimmune prevention. They are also easily harvested with little morbidity, which is an advantage over other mesenchymal stem cell sources.(38)\u0026nbsp;Nevertheless, their ability to proliferate and differentiate into neurogenic lineages such as glial cells (SLCs) is inferior to that of stem cells derived from nervous tissue, so PRP is utilized to accelerate these capabilities in vitro. The therapeutic potential of PRP is attributed to its content of growth factors like TGF-b, PDGF, VEGF, and SDF-1\u0026alpha;, which have been shown to promote neurogenic differentiation and cell proliferation.\u003cspan lang=\"EN-US\"\u003e(28,39)\u003c/span\u003e\u003c/p\u003e\n\u003cp\u003eSchwann-like cells (SLCs) resemble real Schwann cells after being incubated in an induction medium for several days. Most studies have demonstrated the augmentation effect after being implanted in silicon conduit on the peripheral nerve regeneration process. In particular, transplantation of MSCs has been demonstrated to lessen muscle atrophy, promote axon growth and myelination, and minimize inflammation.\u003cspan lang=\"EN-US\"\u003e(40)\u003c/span\u003e\u003c/p\u003e\n\u003cp\u003ePreconditioning MSCs under hypoxic conditions (1%\u0026ndash;7% oxygen), which more closely resemble natural tissue environments than usual normoxic culture (21%), improves their survival through the activation of HIF-1\u0026alpha; and an Akt-dependent pathway. This approach also accelerates the secretion of proangiogenic factors and increases the expression of SDF-1 and its receptors, CXCR4 and CXCR7, thereby improving MSCs engraftment in vivo.\u003cspan lang=\"EN-US\"\u003e(41,42)\u003c/span\u003e Additionally, hypoxic preconditioning boosts the paracrine activity of MSCs and has been successfully applied in treating conditions like myocardial infarction and diabetes-related erectile dysfunction.\u003cspan lang=\"EN-US\"\u003e(43,44)\u003c/span\u003e\u003c/p\u003e\n\u003cp\u003eThe secretome refers to the complex mixture of bioactive molecules secreted by cells into their extracellular milieu. In the context of Schwann-like cells (SLCs) derived from mesenchymal stem cells, especially under hypoxic conditions, the secretome plays a pivotal role in modulating the microenvironment of peripheral nerve injury and accelerating regeneration.(45)\u003c/p\u003e\n\u003cp\u003eUnder hypoxia (typically 1\u0026ndash;5% O₂), SLCs exhibit a shift in paracrine signaling, resulting in upregulation of trophic and immunomodulatory factors. This hypoxia-induced adaptation mimics the physiological environment of injured tissues and primes the cells to secrete a richer, more regenerative profile of factors.(31,46)\u003c/p\u003e\n\u003cp\u003eA multipurpose cytokine, TGF-b\u0026nbsp;is crucial for neuroprotection and peripheral nerve repair. In order to promote nerve regeneration at lesion sites, TGF-b\u0026nbsp; may trigger Schwann cell reprogramming, alter immune cells, increase neuronal growth potential, and control blood-nerve barrier permeability.\u003cspan lang=\"EN-US\"\u003e(47)\u003c/span\u003e Additionally, it stimulates M2 macrophages, which work with Schwann cells to promote axonal regeneration.\u003cspan lang=\"EN-US\"\u003e(48)\u003c/span\u003e\u003csup\u003e\u0026nbsp;\u003c/sup\u003eTGF-b\u0026nbsp;influences Schwann cell proliferation and differentiation, shifting them from a myelinating to an activated state.\u003cspan lang=\"EN-US\"\u003e(49,50)\u003c/span\u003e\u003csup\u003e\u0026nbsp;\u003c/sup\u003eTGF-b\u0026nbsp;may also have extensive biological effects during nerve regeneration through interaction with transcriptional co-regulators and Smad-dependent and -independent signaling.\u003cspan lang=\"EN-US\"\u003e(51,52)\u003c/span\u003e\u003c/p\u003e\n\u003cp\u003eSubsequently, HIF-1\u0026alpha; plays a central role in neuronal regeneration by regulating injury-induced genes and promoting axonal regrowth. It accelerates VEGF expression, which supports nerve repair and angiogenesis. Hypoxia-a crucial condition during peripheral nerve injury-increases HIF-1\u0026alpha; and NGF expression, both of which synergize in regeneration.\u003cspan lang=\"EN-US\"\u003e(53,54)\u003c/span\u003e\u003c/p\u003e\n\u003cp\u003eA myofibroblast marker \u0026alpha;-SMA, is expressed in pericytes and perineurial cells in uninjured nerves, but its role after injury remains unclear.\u003cspan lang=\"EN-US\"\u003e(55)\u003c/span\u003e\u003csup\u003e\u0026nbsp;\u003c/sup\u003eInjury activates fibrotic pathways involving Schwann cells, fibroblasts, and macrophages that clear myelin and may contribute to collagen deposition.\u003cspan lang=\"EN-US\"\u003e(56,57)\u003c/span\u003e\u003csup\u003e\u0026nbsp;\u003c/sup\u003eExcess collagen alters nerve architecture and may hinder regeneration. Various cells-including Schwann cells, fibroblasts, pericytes, and macrophages-synthesize collagen during this process. In addition, endoneurial fibroblasts are also derived from neural crest SCs as well as Schwann cells.\u003cspan lang=\"EN-US\"\u003e(55)\u003c/span\u003e AdMSCs have the effect of downregulating the pro-fibrotic marker \u0026alpha;-SMA gene expression and upregulating the anti-fibrotic fibroblast growth factor and pro-VEGF angiogenic gene.\u003cspan lang=\"EN-US\"\u003e(58)\u003c/span\u003e AdMSCs appear to have differential activity by remodeling scar fibrotic matrix, changing the balance between ECM deposition and degradation for deterioration to occur, even though they come from the same mesodermal layer of myofibroblasts.\u003cspan lang=\"EN-US\"\u003e(58,59)\u003c/span\u003e\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e\u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp;\u0026nbsp;\u003c/strong\u003eNCAM plays an important role during peripheral nerve regeneration. NCAM mediates interactions between axons, and together with cell adhesion molecule L1, it mediates between Schwann cells and axons. Following peripheral nerve injury, NCAM and N-cadherin are upregulated at the proximal ends of nerves. L1 and NCAM were found at the distal end of transected adult peripheral nerves.\u003cspan lang=\"EN-US\"\u003e(12,60)\u003c/span\u003e Moreover, additional research indicates that NCAM/CD56 is crucial for a number of functions, such as adhesion molecules involved in peripheral nerve development and renewal, synaptic plasticity, cognitive function, and myelin formation and maintenance. Axons must be guided toward their target organs by interactions between Schwann cells and regenerated axons via adhesion molecules. Polysialic acid (PSA), an anionic glycan linked to NCAM and present on the surface of Schwann cells and neurons, is one of the important chemicals involved.\u003cspan lang=\"EN-US\"\u003e(61)\u003c/span\u003e\u003c/p\u003e\n\u003cp\u003eCD31, also known as Platelet Endothelial Cell Adhesion Molecule-1 (PECAM-1), is a 130-kDa cell surface marker from the immunoglobulin (Ig) superfamily, expressed in vascular endothelial cells and involved in leukocyte transmigration, anti-apoptotic signaling, and cell adhesion.\u003cspan lang=\"EN-US\"\u003e(62)\u003c/span\u003e Its role in peripheral nerve regeneration is linked to angiogenesis, supported by CD31 immunoreactivity expression patterns, indicating a strong connection between nerve regeneration and vascularization.\u003cspan lang=\"EN-US\"\u003e(63)\u003c/span\u003e\u0026nbsp;\u003c/p\u003e\n\u003cp\u003eNeuN as a marker for post-mitotic neurons, is commonly used to identify neurons after injury or damage. It serves as an indicator of neuronal presence and is considered a surrogate marker for peripheral nerve injury, with increased expression observed in small CGRP-positive DRG neurons during inflammation-suggesting its involvement in specific nociceptive neuron populations.\u003cspan lang=\"EN-US\"\u003e(64)\u003c/span\u003e\u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eLimitation of study\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThis study has several drawbacks, such as not examining other variables involved in the peripheral nerve regeneration process due to acute injury, such as collagen I and II. In addition, this study did not examine the results of peripheral nerve regeneration through histopathological and neurophysiological examinations. Research over a longer period is also needed to obtain a more complete knowledge of the increasing and decreasing levels or expression of several cytokines.\u003c/p\u003e"},{"header":"Conclusions","content":"\u003cp\u003eThis study concluded that hypoxic-conditioned allogeneic SLCs accelerate peripheral nerve regeneration in acute nerve injury, evidenced by increased TGF-β, HIF-1α, NCAM, Neu-N, and CD-31 expression, reduced α-SMA expression, greater axonal density, and improved motor function. These findings highlight key molecular pathways involved in nerve repair and suggest potential for developing targeted therapies. If replicated clinically, hypoxic-conditioned SLCs could offer a novel approach to accelerate nerve regeneration and restore motor function in patients with peripheral nerve injuries.\u003c/p\u003e"},{"header":"Declarations","content":"\u003ch2\u003eAuthor Contribution\u003c/h2\u003e\u003cp\u003eTS and DNU contributed to conceptualization and project administration and took the lead in funding acquisition and investigation.TS and R were responsible for data curation, formal analysis, validation, and visualization.R and HS contributed to methodology and resources.CRSP, DT, HBN, NA, FAR, SR, MFI, and FM contributed to investigation and resources.TS and R supervised the project.TS, R, and HS wrote the original draft of the manuscript.TS, R, DNU, DT, NA, FAR, SR, and MFI reviewed and edited the manuscript.\u0026ldquo;All authors read and approved the final manuscript.\u0026rdquo;\u003c/p\u003e\u003ch2\u003eAcknowledgement\u003c/h2\u003e\u003cp\u003e The authors thank drh Deya Karsari and drh Igo Syaiful Ihsan from the Stem Cell Research and Development Center, Airlangga University, Surabaya, Indonesia, Amed Gatut Guntoro for designing the graphical abstract, and Ms. Agisa Prawesti for assisting in arranging this study, providing technical/laboratory support, and animal care/testing support.\u003c/p\u003e"},{"header":"References","content":"\u003col\u003e\n\u003cli\u003eJou C, Sonti A, Kennemer A, Ramos R, Jack MM, Kwiecien G, et al. Nerve Healing and Future Directions. Operative Techniques in Orthopaedics [Internet]. 2025;35(1):101178. Available from: https://www.sciencedirect.com/science/article/pii/S1048666625000102\u003c/li\u003e\n\u003cli\u003eZhang S, Huang M, Zhi J, Wu S, Wang Y, Pei F. Research Hotspots and Trends of Peripheral Nerve Injuries Based on Web of Science From 2017 to 2021: A Bibliometric Analysis. Frontiers in Neurology. 2022;13(May):1\u0026ndash;12. \u003c/li\u003e\n\u003cli\u003eLi R, Li D hui, Zhang H yu, Wang J, Li X kun, Xiao J. Growth factors-based therapeutic strategies and their underlying signaling mechanisms for peripheral nerve regeneration. Acta Pharmacologica Sinica. 2020;41(10):1289\u0026ndash;300. \u003c/li\u003e\n\u003cli\u003eSuroto H, Antoni I, Siyo A, Steendam TC, Prajasari T, Mulyono HB, et al. Traumatic Brachial Plexus Injury in Indonesia: An Experience from a Developing Country. Journal of Reconstructive Microsurgery. 2021; \u003c/li\u003e\n\u003cli\u003eSumarwoto T, Hadinoto SA, Kaldani F, Aprilya D, Abimanyu DR. The Characteristic of 374 Surgically Treated Traumatic Brachial Plexus Injury Patients at an Indonesian Orthopedic Referral Hospital: An Epidemiologic and Sociodemographic View. Orthopedic Research and Reviews. 2022;14(November):419\u0026ndash;28. \u003c/li\u003e\n\u003cli\u003eRadecka W, Nogalska W, Siemionow M. Peripheral Nerve Protection Strategies : Recent Advances and Potential Clinical Applications. Journal of Functional Biomaterials. 2025;16(153):1\u0026ndash;17. \u003c/li\u003e\n\u003cli\u003eSong X, Li R, Chu X, Li Q, Li R, Li Q, et al. Multilevel analysis of the central\u0026ndash;peripheral\u0026ndash;target organ pathway: contributing to recovery after peripheral nerve injury. Neural Regeneration Research. 2025;20(10):2807\u0026ndash;22. \u003c/li\u003e\n\u003cli\u003eKhaled MM, Ibrahium AM, Abdelgalil AI, El-Saied MA, El-Bably SH. 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Burns \u0026amp; Trauma. 2025;13(May). \u003c/li\u003e\n\u003cli\u003eSharifi M, Kamalabadi-Farahani M, Salehi M, Ebrahimi-Brough S, Alizadeh M. Recent perspectives on the synergy of mesenchymal stem cells with micro/nano strategies in peripheral nerve regeneration-a review. Frontiers in Bioengineering and Biotechnology. 2024;12(July):1\u0026ndash;21. \u003c/li\u003e\n\u003cli\u003eGrosu-Bularda A, Vancea CV, Hodea FV, Cretu A, Bordeanu-Diaconescu EM, Dumitru CS, et al. Optimizing Peripheral Nerve Regeneration: Surgical Techniques, Biomolecular and Regenerative Strategies\u0026mdash;A Narrative Review. International Journal of Molecular Sciences. 2025;26(8):1\u0026ndash;49. \u003c/li\u003e\n\u003cli\u003eFu X, Tong Z, Li QI, Niu Q, Zhang ZHE. Induction of adipose-derived stem cells into Schwann-like cells and observation of Schwann-like cell proliferation. 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Plastic and Reconstructive Surgery. 1989;83(1):129\u0026ndash;36. \u003c/li\u003e\n\u003cli\u003eBrown C, Mackinnon S, Evans P, Bain J, Makino A, Hunter D, et al. Self-Evaluation of Walking-Track Measurement Using a Sciatic Function Index. MICROSURGERY. 1989;10:226\u0026ndash;35. \u003c/li\u003e\n\u003cli\u003eMedinaceli L de, Freed WJ, Wyatt RJ. An Index of the Functional Condition of Rat Sciatic Nerve Based on Measurements Made from Walking Tracks. Experimental neurology. 1982;77:634\u0026ndash;43. \u003c/li\u003e\n\u003cli\u003eMacewan MR, Zellmer ER, Wheeler JJ, Burton H, Moran DW. Regenerated Sciatic Nerve Axons Stimulated through a Chronically Implanted Macro-Sieve Electrode. Frontiers in Neuroscience. 2016;10(December):1\u0026ndash;12. \u003c/li\u003e\n\u003cli\u003eFrese L, Dijkman E, Hoerstrup SP. Adipose Tissue-Derived Stem Cells in Regenerative Medicine. 2016;268\u0026ndash;74. \u003c/li\u003e\n\u003cli\u003eLi H, Han Z, Liu D, Zhao P, Liang S, Xu K. Autologous Platelet-Rich Plasma Promotes Neurogenic Differentiation of Human Adipose-Derived Stem Cells in vitro. International Journal of Neuroscience. 2013;123(3):184\u0026ndash;90. \u003c/li\u003e\n\u003cli\u003eJiang L, Jones S, Jia X. Stem cell transplantation for peripheral nerve regeneration: Current options and opportunities. International Journal of Molecular Sciences. 2017;18(1). \u003c/li\u003e\n\u003cli\u003eSuryawan IGR, Pikir BS, Rantam FA, Ratri AK, Nugraha RA. Hypoxic preconditioning promotes survival of human adipose derived mesenchymal stem cell via expression of prosurvival and proangiogenic biomarkers. F1000Research. 2021;10:843. \u003c/li\u003e\n\u003cli\u003eSumarwoto T, Suroto H, Mahyudin F, Utomo DN, Prijosedjati A, Romaniyanto R, et al. Preconditioning of Hypoxic Culture Increases The Therapeutic Potential of Adipose Derived Mesenchymal Stem Cells Characteristics of MSCs. Open Access Macedonian Journal of Medical Sciences. 2021;9:505\u0026ndash;15. \u003c/li\u003e\n\u003cli\u003eHao D, He C, Ma B, Lankford L, Reynaga L, Farmer DL, et al. Hypoxic Preconditioning Enhances Survival and Proangiogenic Capacity of Human First Trimester Chorionic Villus-Derived Mesenchymal Stem Cells for Fetal Tissue Engineering. Stem Cell International. 2019;2019:1\u0026ndash;12. \u003c/li\u003e\n\u003cli\u003eHu Y, Chen W, Wu L, Jiang L, Qin H, Tang N. Hypoxic preconditioning improves the survival and neural effects of transplanted mesenchymal stem cells via CXCL12/CXCR4 signalling in a rat model of cerebral infarction. Cell Biochemistry and Function. 2019;37(7):504\u0026ndash;15. \u003c/li\u003e\n\u003cli\u003eDaneshmandi L, Shah S, Jafari T, Bhattacharjee M, Momah D, Saveh-Shemshaki N, et al. Emergence of the Stem Cell Secretome in Regenerative Engineering. Trends in Biotechnology. 2020;38(12):1373\u0026ndash;84. \u003c/li\u003e\n\u003cli\u003eV\u0026eacute;lez ICV, Dom\u0026iacute;nguez CMC, S\u0026aacute;nchez MJF, Garavito-aguilar ZV. Hypoxia and Tissue Regeneration: Adaptive Mechanisms and Therapeutic Opportunities. International Journal of Molecular Sciences. 2025;26(9272):1\u0026ndash;43. \u003c/li\u003e\n\u003cli\u003eYe Z, Wei J, Zhan C, Hou J. Role of Transforming Growth Factor Beta in Peripheral Nerve Regeneration : Cellular and Molecular Mechanisms. Frontiers in Neuroscience. 2022;16(June):1\u0026ndash;16. \u003c/li\u003e\n\u003cli\u003eHealy LM, Perron G, Won S, Rezk A, Ludwin SK, Moore CS, et al. MerTK Is a Functional Regulator of Myelin Phagocytosis by Human Myeloid Cells. The Journal of Immunology. 2016;196(9 March 2016):1\u0026ndash;11. \u003c/li\u003e\n\u003cli\u003eJeon K, Huxlin KR. How scars shape the neural landscape : Key molecular mediators of TGF- \u0026beta; 1 \u0026rsquo; s anti- neuritogenic effects. PLoS ONE [Internet]. 2020;15(11)(November 24, 2020):1\u0026ndash;19. Available from: http://dx.doi.org/10.1371/journal.pone.0234950\u003c/li\u003e\n\u003cli\u003eClements MP, Byrne E, Guerrero LFC, Lloyd AC, Marguerat S, Clements MP, et al. The Wound Microenvironment Reprograms Schwann Cells to Invasive Mesenchymal-like Cells to Drive Article The Wound Microenvironment Reprograms Schwann Cells to Invasive Mesenchymal-like Cells to Drive Peripheral Nerve Regeneration. Neuron. 2017;96(September 27, 2017):98\u0026ndash;114. \u003c/li\u003e\n\u003cli\u003eDeng Z, Fan T, Xiao C, Tian H, Zheng Y, Li C, et al. TGF-\u0026beta; signaling in health, disease, and therapeutics. Signal Transduction and Targeted Therapy [Internet]. 2024;9(1). Available from: http://dx.doi.org/10.1038/s41392-024-01764-w\u003c/li\u003e\n\u003cli\u003eLi S, Gu X, Yi S. The regulatory effects of transforming growth factor-\u0026beta; on nerve regeneration. Cell Transplantation. 2017;26(3):381\u0026ndash;94. \u003c/li\u003e\n\u003cli\u003eAn S, Zhou M, Li Z, Feng M, Cao G, Lu S, et al. Administration of CoCl 2 Improves Functional Recovery in a Rat Model of Sciatic Nerve Transection Injury. Int J Med Sci. 2018;15(13):1423\u0026ndash;32. \u003c/li\u003e\n\u003cli\u003eCho Y, Shin JE, Ewan EE, Oh YM, Pita-thomas W, Cavalli V. Activating Injury-Responsive Genes with Hypoxia Enhances Axon Regeneration through Neuronal Article Activating Injury-Responsive Genes with Hypoxia Enhances Axon Regeneration through Neuronal HIF-1 a. Neuron [Internet]. 2015;88(November 18, 2015):1\u0026ndash;15. Available from: http://dx.doi.org/10.1016/j.neuron.2015.09.050\u003c/li\u003e\n\u003cli\u003eRivlin M, Miller A, Tulipan J, Beredjiklian PK, Wang ML, Fertala J, et al. Patterns of production of collagen- \u0026shy; rich deposits in peripheral nerves in response to injury : A pilot study in a rabbit model. Brain and Behaviour. 2017;(September 2016):1\u0026ndash;10. \u003c/li\u003e\n\u003cli\u003eChen P, Cescon M, Megighian A, Bonaldo P. Collagen VI regulates peripheral nerve myelination and function. The FASEB Journal. 2014;28(March 2014):1145\u0026ndash;56. \u003c/li\u003e\n\u003cli\u003eKendall RT, Feghali-Bostwick CA. Fibroblasts in fibrosis: Novel roles and mediators. Frontiers in Pharmacology. 2014;5 MAY(May):1\u0026ndash;13. \u003c/li\u003e\n\u003cli\u003eVanderstichele S, Vranckx JJ. Anti-fibrotic effect of adipose-derived stem cells on fobrotic scars. World Journal of Stem Cells [Internet]. 2021;14(2):200\u0026ndash;13. Available from: https://www.wjgnet.com/1948-0210/full/v14/i2/200.htm\u003c/li\u003e\n\u003cli\u003eKlingberg F, Hinz B, White ES. The myofibroblast matrix: implications for tissue repair and fibrosis. J Pathol. 2013;229(2):298\u0026ndash;309. \u003c/li\u003e\n\u003cli\u003eWeledji EP, Assob JC. The ubiquitous neural cell adhesion molecule ( N-CAM ). Annals of Medicine and Surgery [Internet]. 2014;3(3):77\u0026ndash;81. Available from: http://dx.doi.org/10.1016/j.amsu.2014.06.014\u003c/li\u003e\n\u003cli\u003eBolivar S, Navarro X, Udina E. Schwann Cell Role in Selectivity of Nerve Regeneration. Cells. 2020;9(2131):1\u0026ndash;18. \u003c/li\u003e\n\u003cli\u003eLi Y, Zhang Z, Kim H, Han S, Kim S. Molecular and Cellular Neuroscience CD31 + cell transplantation promotes recovery from peripheral neuropathy. Molecular and Cellular Neuroscience [Internet]. 2014;62:60\u0026ndash;7. Available from: http://dx.doi.org/10.1016/j.mcn.2014.08.005\u003c/li\u003e\n\u003cli\u003eD\u0026ouml;mer P, Kayal J, Bienhold UJ, Kewitz B, Kretschmer T, Heinen C. Rapid and efficient immunomagnetic isolation of endothelial cells from human peripheral nerves. Scientific Reports [Internet]. 2021;11(1951):1\u0026ndash;10. Available from: https://doi.org/10.1038/s41598-021-81361-x\u003c/li\u003e\n\u003cli\u003eAnderson MB, Das S, Miller KE. Subcellular localization of neuronal nuclei (NeuN) antigen in size and calcitonin gene-related peptide (CGRP) populations of dorsal root ganglion (DRG) neurons during acute peripheral inflammation. Neurosci Lett. 2021;760:1\u0026ndash;21.\u003c/li\u003e\n\u003c/ol\u003e"},{"header":"Tables","content":"\u003cp\u003e\u003cstrong\u003eTable 1\u0026nbsp;\u003c/strong\u003eDifferences in TGF-b, VEGF, \u0026alpha;-SMA, CD-31, NCAM, and Neu-N values between the two intervention groups (hypoxic conditioned allogeneic Schwann-like cells and secretome) and the control group at weeks three and six.\u003c/p\u003e\n\u003ctable border=\"1\" cellspacing=\"0\" cellpadding=\"0\" align=\"\"\u003e\n \u003ctbody\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\" style=\"width: 75px;\"\u003e\n \u003cp\u003e\u003cstrong\u003e\u0026nbsp;\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 107px;\"\u003e\n \u003cp\u003e\u003cstrong\u003eTGF-\u003c/strong\u003e\u003cstrong\u003eb\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 101px;\"\u003e\n \u003cp\u003e\u003cstrong\u003eVEGF\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 76px;\"\u003e\n \u003cp\u003e\u003cstrong\u003e\u0026alpha;-SMA\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 76px;\"\u003e\n \u003cp\u003e\u003cstrong\u003eCD-31\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 76px;\"\u003e\n \u003cp\u003e\u003cstrong\u003eNCAM\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 90px;\"\u003e\n \u003cp\u003e\u003cstrong\u003eNeu-N\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd colspan=\"7\" style=\"width: 602px;\"\u003e\n \u003cp\u003e\u003cstrong\u003eWeek 3\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd style=\"width: 75px;\"\u003e\n \u003cp\u003eControl\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 107px;\"\u003e\n \u003cp\u003e37,67\u0026plusmn;2,31\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 101px;\"\u003e\n \u003cp\u003e151,67\u0026plusmn;48,60\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 76px;\"\u003e\n \u003cp\u003e29,94\u0026plusmn;3,27\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 76px;\"\u003e\n \u003cp\u003e26,57\u0026plusmn;4,28\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 76px;\"\u003e\n \u003cp\u003e36,57\u0026plusmn;4,96\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 90px;\"\u003e\n \u003cp\u003e76,16\u0026plusmn;15,62\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd style=\"width: 75px;\"\u003e\n \u003cp\u003eIntervention 1\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 107px;\"\u003e\n \u003cp\u003e113,60\u0026plusmn;14,03\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 101px;\"\u003e\n \u003cp\u003e203,00\u0026plusmn;112,46\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 76px;\"\u003e\n \u003cp\u003e18,70\u0026plusmn;2,56\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 76px;\"\u003e\n \u003cp\u003e25,01\u0026plusmn;2,44\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 76px;\"\u003e\n \u003cp\u003e63,01\u0026plusmn;3,46\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 90px;\"\u003e\n \u003cp\u003e104,30\u0026plusmn;7,25\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd style=\"width: 75px;\"\u003e\n \u003cp\u003eIntervention 2\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 107px;\"\u003e\n \u003cp\u003e130,33\u0026plusmn;24,69\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 101px;\"\u003e\n \u003cp\u003e201,67\u0026plusmn;31,79\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 76px;\"\u003e\n \u003cp\u003e14,09\u0026plusmn;1,48\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 76px;\"\u003e\n \u003cp\u003e35,88\u0026plusmn;2,25\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 76px;\"\u003e\n \u003cp\u003e52,64\u0026plusmn;5,96\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 90px;\"\u003e\n \u003cp\u003e74,80\u0026plusmn;1,90\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd style=\"width: 75px;\"\u003e\n \u003cp\u003ep-value\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 107px;\"\u003e\n \u003cp\u003e0.002d*\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 101px;\"\u003e\n \u003cp\u003e0.235d\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 76px;\"\u003e\n \u003cp\u003e0.000c*\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 76px;\"\u003e\n \u003cp\u003e0.003c*\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 76px;\"\u003e\n \u003cp\u003e0.000c*\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 90px;\"\u003e\n \u003cp\u003e0.049e*\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd colspan=\"7\" style=\"width: 602px;\"\u003e\n \u003cp\u003e\u003cstrong\u003eWeek 6\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd style=\"width: 75px;\"\u003e\n \u003cp\u003eControl\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 107px;\"\u003e\n \u003cp\u003e69,33\u0026plusmn;5,51\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 101px;\"\u003e\n \u003cp\u003e220,33\u0026plusmn;13,05\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 76px;\"\u003e\n \u003cp\u003e30,07\u0026plusmn;4,09\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 76px;\"\u003e\n \u003cp\u003e27,54\u0026plusmn;1,30\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 76px;\"\u003e\n \u003cp\u003e49,14\u0026plusmn;3,32\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 90px;\"\u003e\n \u003cp\u003e87,71\u0026plusmn;5,66\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd style=\"width: 75px;\"\u003e\n \u003cp\u003eIntervention 1\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 107px;\"\u003e\n \u003cp\u003e172,80\u0026plusmn;11,76\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 101px;\"\u003e\n \u003cp\u003e239,00\u0026plusmn;41,89\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 76px;\"\u003e\n \u003cp\u003e22,15\u0026plusmn;1,87\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 76px;\"\u003e\n \u003cp\u003e34,64\u0026plusmn;3,66\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 76px;\"\u003e\n \u003cp\u003e77,20\u0026plusmn;5,39\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 90px;\"\u003e\n \u003cp\u003e166,41\u0026plusmn;10,85\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd style=\"width: 75px;\"\u003e\n \u003cp\u003eIntervention 2\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 107px;\"\u003e\n \u003cp\u003e196,00\u0026plusmn;10,58\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 101px;\"\u003e\n \u003cp\u003e297,33\u0026plusmn;25,11\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 76px;\"\u003e\n \u003cp\u003e21,52\u0026plusmn;2,78\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 76px;\"\u003e\n \u003cp\u003e40,63\u0026plusmn;1,87\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 76px;\"\u003e\n \u003cp\u003e73,93\u0026plusmn;3,91\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 90px;\"\u003e\n \u003cp\u003e127,54\u0026plusmn;12,21\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd style=\"width: 75px;\"\u003e\n \u003cp\u003ep-value\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 107px;\"\u003e\n \u003cp\u003e0.000c*\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 101px;\"\u003e\n \u003cp\u003e0.323c\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 76px;\"\u003e\n \u003cp\u003e0.003c*\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 76px;\"\u003e\n \u003cp\u003e0.000c*\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 76px;\"\u003e\n \u003cp\u003e0.000c*\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 90px;\"\u003e\n \u003cp\u003e0.000c*\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd colspan=\"7\" valign=\"top\" style=\"width: 602px;\"\u003e\n \u003cp\u003e\u003csup\u003ea\u003c/sup\u003eIndependent T test \u0026nbsp; \u003csup\u003eb\u003c/sup\u003eMann-Whithney \u0026nbsp;\u003csup\u003ec\u003c/sup\u003eAnova test \u0026nbsp;\u003csup\u003ed\u003c/sup\u003eKruskal Wallis \u0026nbsp;\u003csup\u003ee\u003c/sup\u003eBrown-Forsythe \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp;\u0026nbsp;\u003c/p\u003e\n \u003cp\u003e*p considered statistically significant\u003c/p\u003e\n \u003cp\u003eIntervention 1 = SLCs \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; Intervention 2 = secretome SLCs\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003c/tbody\u003e\n\u003c/table\u003e\n\u003cp\u003e\u003cstrong\u003e\u0026nbsp;\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eTable 2\u0026nbsp;\u003c/strong\u003eDifferences in TGF-b, VEGF, \u0026alpha;-SMA, CD-31, NCAM, and Neu-N values between week three and six in the two intervention groups (hypoxic conditioned allogeneic Schwann-like cells and secretome) and the control group.\u0026nbsp;\u003c/p\u003e\n\u003ctable border=\"1\" cellspacing=\"0\" cellpadding=\"0\" align=\"\" width=\"611\"\u003e\n \u003ctbody\u003e\n \u003ctr\u003e\n \u003ctd style=\"width: 56px;\"\u003e\n \u003cp\u003e\u003cstrong\u003e\u0026nbsp;\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 100px;\"\u003e\n \u003cp\u003e\u003cstrong\u003eTGF-\u003c/strong\u003e\u003cstrong\u003eb\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 104px;\"\u003e\n \u003cp\u003e\u003cstrong\u003eVEGF\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 88px;\"\u003e\n \u003cp\u003e\u003cstrong\u003e\u0026alpha;-SMA\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 75px;\"\u003e\n \u003cp\u003e\u003cstrong\u003eCD-31\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 92px;\"\u003e\n \u003cp\u003e\u003cstrong\u003eNCAM\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 94px;\"\u003e\n \u003cp\u003e\u003cstrong\u003eNeu-N\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 0px;\"\u003e\n \u003cp\u003e\u0026nbsp;\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd colspan=\"8\" style=\"width: 611px;\"\u003e\n \u003cp\u003e\u003cstrong\u003eControl Group\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd style=\"width: 56px;\"\u003e\n \u003cp\u003eWeek 3\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 100px;\"\u003e\n \u003cp\u003e37,67\u0026plusmn;2,31\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 104px;\"\u003e\n \u003cp\u003e151,67\u0026plusmn;48,60\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 88px;\"\u003e\n \u003cp\u003e29,94\u0026plusmn;3,27\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 75px;\"\u003e\n \u003cp\u003e26,57\u0026plusmn;4,28\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 92px;\"\u003e\n \u003cp\u003e36,57\u0026plusmn;4,96\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 94px;\"\u003e\n \u003cp\u003e76,16\u0026plusmn;15,62\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 0px;\"\u003e\n \u003cp\u003e\u0026nbsp;\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd style=\"width: 56px;\"\u003e\n \u003cp\u003eWeek 6\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 100px;\"\u003e\n \u003cp\u003e69,33\u0026plusmn;5,51\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 104px;\"\u003e\n \u003cp\u003e220,33\u0026plusmn;13,05\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 88px;\"\u003e\n \u003cp\u003e30,07\u0026plusmn;4,09\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 75px;\"\u003e\n \u003cp\u003e27,54\u0026plusmn;1,30\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 92px;\"\u003e\n \u003cp\u003e49,14\u0026plusmn;3,32\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 94px;\"\u003e\n \u003cp\u003e87,71\u0026plusmn;5,66\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 0px;\"\u003e\n \u003cp\u003e\u0026nbsp;\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd style=\"width: 56px;\"\u003e\n \u003cp\u003ep-value\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 100px;\"\u003e\n \u003cp\u003e0.002a*\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 104px;\"\u003e\n \u003cp\u003e0.043b*\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 88px;\"\u003e\n \u003cp\u003e0.966b\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 75px;\"\u003e\n \u003cp\u003e0.615b\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 92px;\"\u003e\n \u003cp\u003e0.010b*\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 94px;\"\u003e\n \u003cp\u003e0.168b\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 0px;\"\u003e\n \u003cp\u003e\u0026nbsp;\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd colspan=\"8\" style=\"width: 611px;\"\u003e\n \u003cp\u003e\u003cstrong\u003eIntervention 1 Group\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\" style=\"width: 56px;\"\u003e\n \u003cp\u003eWeek 3\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 100px;\"\u003e\n \u003cp\u003e113,60\u0026plusmn;14,03\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 104px;\"\u003e\n \u003cp\u003e203,00\u0026plusmn;112,46\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 88px;\"\u003e\n \u003cp\u003e18,70\u0026plusmn;2,56\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 75px;\"\u003e\n \u003cp\u003e25,01\u0026plusmn;2,44\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 92px;\"\u003e\n \u003cp\u003e63,01\u0026plusmn;3,46\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 94px;\"\u003e\n \u003cp\u003e104,30\u0026plusmn;7,25\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 0px;\"\u003e\n \u003cp\u003e\u0026nbsp;\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\" style=\"width: 56px;\"\u003e\n \u003cp\u003eWeek 6\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 100px;\"\u003e\n \u003cp\u003e172,80\u0026plusmn;11,76\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 104px;\"\u003e\n \u003cp\u003e239,00\u0026plusmn;41,89\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 88px;\"\u003e\n \u003cp\u003e22,15\u0026plusmn;1,87\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 75px;\"\u003e\n \u003cp\u003e34,64\u0026plusmn;3,66\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 92px;\"\u003e\n \u003cp\u003e77,20\u0026plusmn;5,39\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 94px;\"\u003e\n \u003cp\u003e166,41\u0026plusmn;10,85\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 0px;\"\u003e\n \u003cp\u003e\u0026nbsp;\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\" style=\"width: 56px;\"\u003e\n \u003cp\u003ep-value\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 100px;\"\u003e\n \u003cp\u003e0.002a*\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 104px;\"\u003e\n \u003cp\u003e0.065a\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 88px;\"\u003e\n \u003cp\u003e0.030b*\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 75px;\"\u003e\n \u003cp\u003e0.001b*\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 92px;\"\u003e\n \u003cp\u003e0.001b*\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 94px;\"\u003e\n \u003cp\u003e0.000b*\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 0px;\"\u003e\n \u003cp\u003e\u0026nbsp;\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd colspan=\"8\" style=\"width: 611px;\"\u003e\n \u003cp\u003e\u003cstrong\u003eIntervention 2 Group\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\" style=\"width: 56px;\"\u003e\n \u003cp\u003eWeek 3\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 100px;\"\u003e\n \u003cp\u003e130,33\u0026plusmn;24,69\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 104px;\"\u003e\n \u003cp\u003e201,67\u0026plusmn;31,79\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 88px;\"\u003e\n \u003cp\u003e14,09\u0026plusmn;1,48\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 75px;\"\u003e\n \u003cp\u003e35,88\u0026plusmn;2,25\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 92px;\"\u003e\n \u003cp\u003e52,64\u0026plusmn;5,96\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 94px;\"\u003e\n \u003cp\u003e74,80\u0026plusmn;1,90\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 0px;\"\u003e\n \u003cp\u003e\u0026nbsp;\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\" style=\"width: 56px;\"\u003e\n \u003cp\u003eWeek 6\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 100px;\"\u003e\n \u003cp\u003e196,00\u0026plusmn;10,58\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 104px;\"\u003e\n \u003cp\u003e297,33\u0026plusmn;25,11\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 88px;\"\u003e\n \u003cp\u003e21,52\u0026plusmn;2,78\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 75px;\"\u003e\n \u003cp\u003e40,63\u0026plusmn;1,87\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 92px;\"\u003e\n \u003cp\u003e73,93\u0026plusmn;3,91\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 94px;\"\u003e\n \u003cp\u003e127,54\u0026plusmn;12,21\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 0px;\"\u003e\n \u003cp\u003e\u0026nbsp;\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\" style=\"width: 56px;\"\u003e\n \u003cp\u003ep-value\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 100px;\"\u003e\n \u003cp\u003e0.001b*\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 104px;\"\u003e\n \u003cp\u003e0.008b*\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 88px;\"\u003e\n \u003cp\u003e0.015b*\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 75px;\"\u003e\n \u003cp\u003e0.048b*\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 92px;\"\u003e\n \u003cp\u003e0.007b*\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 94px;\"\u003e\n \u003cp\u003e0.002b*\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 0px;\"\u003e\n \u003cp\u003e\u0026nbsp;\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd colspan=\"8\" style=\"width: 611px;\"\u003e\n \u003cp\u003e\u003cstrong\u003e\u0026nbsp; \u0026nbsp; \u0026nbsp;\u003c/strong\u003e\u003csup\u003ea\u003c/sup\u003eMann-Whithney and \u003csup\u003eb\u003c/sup\u003eIndependent T-test were performed;\u003c/p\u003e\n \u003cp\u003e*p considered statistically significant.\u003c/p\u003e\n \u003cp\u003eIntervention 1 = SLCs \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; Intervention 2 = secretome SLCs\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003c/tbody\u003e\n\u003c/table\u003e\n\u003cp\u003e\u003cbr\u003e\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eTable 3\u0026nbsp;\u003c/strong\u003eDifferences in sciatic function index between the two intervention groups (hypoxic conditioned allogeneic Schwann-like cells and secretome) and the control group at pre-intervention and weeks one to six.\u003c/p\u003e\n\u003ctable border=\"1\" cellspacing=\"0\" cellpadding=\"0\" align=\"\" width=\"652\"\u003e\n \u003ctbody\u003e\n \u003ctr\u003e\n \u003ctd style=\"width: 56px;\"\u003e\n \u003cp\u003e\u003cstrong\u003e\u0026nbsp;\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 85px;\"\u003e\n \u003cp\u003e\u003cstrong\u003ePre\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 79px;\"\u003e\n \u003cp\u003e\u003cstrong\u003eWeek 1\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 91px;\"\u003e\n \u003cp\u003e\u003cstrong\u003eWeek 2\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 85px;\"\u003e\n \u003cp\u003e\u003cstrong\u003eWeek 3\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 85px;\"\u003e\n \u003cp\u003e\u003cstrong\u003eWeek 4\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 85px;\"\u003e\n \u003cp\u003e\u003cstrong\u003eWeek 5\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 85px;\"\u003e\n \u003cp\u003e\u003cstrong\u003eWeek 6\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 0px;\"\u003e\n \u003cp\u003e\u0026nbsp;\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd style=\"width: 56px;\"\u003e\n \u003cp\u003eControl\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 85px;\"\u003e\n \u003cp\u003e-7,79\u0026plusmn;2,37\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 79px;\"\u003e\n \u003cp\u003e-73,27\u0026plusmn;7,76\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 91px;\"\u003e\n \u003cp\u003e-66,82\u0026plusmn;5,05\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 85px;\"\u003e\n \u003cp\u003e-60,49\u0026plusmn;7,86\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 85px;\"\u003e\n \u003cp\u003e-56,43\u0026plusmn;8,02\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 85px;\"\u003e\n \u003cp\u003e-54,99\u0026plusmn;7,53\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 85px;\"\u003e\n \u003cp\u003e-38,47\u0026plusmn;5,79\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 0px;\"\u003e\n \u003cp\u003e\u0026nbsp;\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd style=\"width: 56px;\"\u003e\n \u003cp\u003eIntervention 1\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 85px;\"\u003e\n \u003cp\u003e-11,27\u0026plusmn;4,43\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 79px;\"\u003e\n \u003cp\u003e-70,4\u0026plusmn;6,57\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 91px;\"\u003e\n \u003cp\u003e-60,12\u0026plusmn;4,01\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 85px;\"\u003e\n \u003cp\u003e-49,37\u0026plusmn;7,12\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 85px;\"\u003e\n \u003cp\u003e-44,6\u0026plusmn;4,89\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 85px;\"\u003e\n \u003cp\u003e-37,83\u0026plusmn;4,49\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 85px;\"\u003e\n \u003cp\u003e-23,13\u0026plusmn;3,23\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 0px;\"\u003e\n \u003cp\u003e\u0026nbsp;\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd style=\"width: 56px;\"\u003e\n \u003cp\u003eIntervention 2\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 85px;\"\u003e\n \u003cp\u003e-8,45\u0026plusmn;1,82\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 79px;\"\u003e\n \u003cp\u003e-64,52\u0026plusmn;6,2\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 91px;\"\u003e\n \u003cp\u003e-50,01\u0026plusmn;11,52\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 85px;\"\u003e\n \u003cp\u003e-31,91\u0026plusmn;7,11\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 85px;\"\u003e\n \u003cp\u003e-25,07\u0026plusmn;3,1\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 85px;\"\u003e\n \u003cp\u003e-19,74\u0026plusmn;0,69\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 85px;\"\u003e\n \u003cp\u003e-15,18\u0026plusmn;2,5\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 0px;\"\u003e\n \u003cp\u003e\u0026nbsp;\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd style=\"width: 56px;\"\u003e\n \u003cp\u003ep-value\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 85px;\"\u003e\n \u003cp\u003e0.091a\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 79px;\"\u003e\n \u003cp\u003e0.341a\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 91px;\"\u003e\n \u003cp\u003e0.006b*\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 85px;\"\u003e\n \u003cp\u003e0.001a*\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 85px;\"\u003e\n \u003cp\u003e0.012a*\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 85px;\"\u003e\n \u003cp\u003e0.001a*\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 85px;\"\u003e\n \u003cp\u003e0.004b*\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 0px;\"\u003e\n \u003cp\u003e\u0026nbsp;\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd colspan=\"9\" valign=\"top\" style=\"width: 652px;\"\u003e\n \u003cp\u003e\u003csup\u003ea\u003c/sup\u003eIndependent T-test and \u003csup\u003eb\u003c/sup\u003eMann-Whitney were performed;\u003c/p\u003e\n \u003cp\u003e*p considered statistically significant.\u003c/p\u003e\n \u003cp\u003eIntervention 1 = SLCs \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; Intervention 2 = secretome SLCs\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003c/tbody\u003e\n\u003c/table\u003e\n\u003cp\u003e\u0026nbsp;\u003c/p\u003e"}],"fulltextSource":"","fullText":"","funders":[],"hasAdminPriorityOnWorkflow":false,"hasManuscriptDocX":true,"hasOptedInToPreprint":true,"hasPassedJournalQc":"","hasAnyPriority":false,"hideJournal":false,"highlight":"","institution":"","isAcceptedByJournal":false,"isAuthorSuppliedPdf":false,"isDeskRejected":"","isHiddenFromSearch":false,"isInQc":false,"isInWorkflow":false,"isPdf":false,"isPdfUpToDate":true,"isWithdrawnOrRetracted":false,"journal":{"display":true,"email":"[email protected]","identity":"stem-cell-research-and-therapy","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":false,"externalIdentity":"scrt","sideBox":"Learn more about [Stem Cell Research \u0026 Therapy](http://stemcellres.biomedcentral.com)","snPcode":"","submissionUrl":"https://www.editorialmanager.com/scrt/default.aspx","title":"Stem Cell Research \u0026 Therapy","twitterHandle":"@BioMedCentral","acdcEnabled":true,"dfaEnabled":true,"editorialSystem":"em","reportingPortfolio":"BMC/SO AJ","inReviewEnabled":true,"inReviewRevisionsEnabled":true},"keywords":"Hypoxic conditioned Schwann-like cells (SLCs), Peripheral nerve regeneration, Acute peripheral nerve injury (PNI), Adipose-derived mesenchymal stem cells (AdMSCs), Secretome, In-vivo rat model","lastPublishedDoi":"10.21203/rs.3.rs-8423634/v1","lastPublishedDoiUrl":"https://doi.org/10.21203/rs.3.rs-8423634/v1","license":{"name":"CC BY 4.0","url":"https://creativecommons.org/licenses/by/4.0/"},"manuscriptAbstract":"\u003cp\u003e\u003cstrong\u003eBackground: \u003c/strong\u003eThis study investigates nerve regeneration augmentation using hypoxic allogeneic Schwann-like cells by analyzing HIF-1α, CD-31, Neu-N, α-SMA, NCAM, TGF-β, VEGF, and motor function.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eMethods\u003c/strong\u003e: This in-vivo study on Rattus norvegicus Wistar divided subjects into intervention (suture plus hypoxic allogeneic SLCs) and control (suture only) groups. SLCs were derived from Adipose Mesenchymal Stem Cells using Kingham's protocol with 10% PRP and 1% hypoxia. ELISA, IHC, rt-PCR were done at weeks 3 and 6, and walking track analysis with sciatic function index (SFI) was performed from weeks 0 to 6.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eResults: \u003c/strong\u003eThe intervention group expressed HIF-1αmore clearly, especially in week 6. In addition, there were statistically significant differences in TGF-b(p=0.002), α-SMA (p=0.000), NCAM (p=0.000), and Neu-N (p=0.049) at week 3, as well as TGF-b (p=0.000), α-SMA (p=0.003), CD-31 (p=0.000), NCAM (p=0.000), and Neu-N (p=0.000) at week 6 between interventions and control group. Significant differences were also found in TGF-b, α-SMA, CD-31, NCAM, and Neu-N between weeks 3 and 6 in the intervention group. Furthermore, differences were also found in the sciatic function index at weeks 2 to 6 (p\u0026lt;0.050) between the intervention group and the control group.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eConclusion: \u003c/strong\u003eAdministration of hypoxic-conditioned allogeneic SLCs accelerated peripheral nerve regeneration in acute peripheral nerve injury (PNI), as evidenced by increased TGF-blevels, HIF-1α and NCAM expression, the axonal density of peripheral nerves through the expression of NeuN protein, and the number of capillaries through expression of CD-31; decreased expression of α-SMA; and improved motor function.\u003c/p\u003e","manuscriptTitle":"Augmentation of peripheral nerve regeneration by hypoxic allogenic Schwann-like cells in acute nerve injury of Rattus norvegicus","msid":"","msnumber":"","nonDraftVersions":[{"code":1,"date":"2026-02-13 08:00:30","doi":"10.21203/rs.3.rs-8423634/v1","editorialEvents":[{"type":"communityComments","content":0},{"type":"decision","content":"Revision requested","date":"2026-02-11T18:42:22+00:00","index":"","fulltext":""},{"type":"editorInvitedReview","content":"","date":"2026-02-11T17:44:44+00:00","index":"hide","fulltext":""},{"type":"editorInvitedReview","content":"","date":"2026-02-10T23:34:44+00:00","index":"hide","fulltext":""},{"type":"reviewerAgreed","content":"241121953294906932830064096433036056507","date":"2026-02-10T04:15:48+00:00","index":"hide","fulltext":""},{"type":"reviewerAgreed","content":"336382289393181447127052302995397760331","date":"2026-02-09T14:10:35+00:00","index":"hide","fulltext":""},{"type":"reviewersInvited","content":"","date":"2026-02-07T16:17:00+00:00","index":"","fulltext":""},{"type":"editorAssigned","content":"","date":"2026-01-30T14:37:37+00:00","index":"","fulltext":""},{"type":"checksComplete","content":"","date":"2026-01-20T01:52:19+00:00","index":"","fulltext":""},{"type":"submitted","content":"Stem Cell Research \u0026 Therapy","date":"2026-01-19T02:17:56+00:00","index":"","fulltext":""}],"status":"published","journal":{"display":true,"email":"[email protected]","identity":"stem-cell-research-and-therapy","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":false,"externalIdentity":"scrt","sideBox":"Learn more about [Stem Cell Research \u0026 Therapy](http://stemcellres.biomedcentral.com)","snPcode":"","submissionUrl":"https://www.editorialmanager.com/scrt/default.aspx","title":"Stem Cell Research \u0026 Therapy","twitterHandle":"@BioMedCentral","acdcEnabled":true,"dfaEnabled":true,"editorialSystem":"em","reportingPortfolio":"BMC/SO AJ","inReviewEnabled":true,"inReviewRevisionsEnabled":true}}],"origin":"","ownerIdentity":"77a403c3-2255-49b6-a8bf-e2ca5aed6bda","owner":[],"postedDate":"February 13th, 2026","published":true,"recentEditorialEvents":[],"rejectedJournal":[],"revision":"","amendment":"","status":"under-review","subjectAreas":[],"tags":[],"updatedAt":"2026-03-16T08:25:24+00:00","versionOfRecord":[],"versionCreatedAt":"2026-02-13 08:00:30","video":"","vorDoi":"","vorDoiUrl":"","workflowStages":[]},"version":"v1","identity":"rs-8423634","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-8423634","identity":"rs-8423634","version":["v1"]},"buildId":"XKTyCvWXoU3ODBz1xrDgd","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}