Direct Lentiviral-Cyclooxygenase 2 Application to the Tendon-Bone Interface Promotes Osteointegration and Enhances Return of the Pull-Out Tensile Strength of the Tendon Graft in a Rat Model of Biceps Tenodesis

This study sought to determine if direct application of the lentiviral (LV)-cyclooxygenase 2 (COX2) vector to the tendon-bone interface would promote osteointegration of the tendon graft in a rat model of biceps tenodesis. The LV-COX2 gene transfer strategy was chosen for investigation because a similar COX2 gene transfer strategy promoted bony bridging of the fracture gap during bone repair, which involves similar histologic transitions that occur in osteointegration. Briefly, a 1.14-mm diameter tunnel was drilled in the mid-groove of the humerus of adult Fischer 344 rats. The LV-COX2 or βgal control vector was applied directly into the bone tunnel and onto the end of the tendon graft, which was then pulled into the bone tunnel. A poly-L-lactide pin was press-fitted into the tunnel as interference fixation. Animals were sacrificed at 3, 5, or 8 weeks for histology analysis of osteointegration. The LV-COX2 gene transfer strategy enhanced neo-chondrogenesis at the tendon-bone interface but with only marginal effect on de novo bone formation. The tendon-bone interface of the LV-COX2-treated tenodesis showed the well-defined tendon-to-fibrocartilage-to-bone histologic transitions that are indicative of osteointegration of the tendon graft. The LV-COX2 in vivo gene transfer strategy also significantly enhanced angiogenesis at the tendon-bone interface. To determine if the increased osteointegration was translated into an improved pull-out mechanical strength property, the pull-out tensile strength of the LV-COX2-treated tendon grafts was determined with a pull-out mechanical testing assay. The LV-COX2 strategy yielded a significant improvement in the return of the pull-out strength of the tendon graft after 8 weeks. In conclusion, the COX2-based in vivo gene transfer strategy enhanced angiogenesis, osteointegration and improved return of the pull-out strength of the tendon graft. Thus, this strategy has great potential to be developed into an effective therapy to promote tendon-to-bone healing after tenodesis or related surgeries.     

Enhancement of tendon-bone healing with the use of bone morphogenetic protein-2 inserted into the suture anchor hole in a rabbit patellar tendon model

Background aimsSuture anchor fixation failure has been reported as a result of anchor loosening and migration during the tendon-bone repair. The aim of this study was to evaluate the effects of bone morphogenetic protein-2 (BMP-2) inserted into the suture anchor hole on bone formation and the tendon-bone healing.MethodsBoth back legs of 24 New Zealand White rabbits (n = 48) were used in this study. A metal suture anchor was then placed 5 mm below the cortex. In the control group, the space over the eyelet of the anchor (suture anchor hole) was not filled. In the experimental group, the suture anchor hole was filled with 0.1 mL of fibrin glue (group 2) or collagen gel (group 3) with 1 μg BMP-2. Histologic analysis, real-time-polymerase chain reaction, bone density and failure load measurement were performed, and differences were analyzed at 4 and 8 weeks.ResultsHistologic analysis revealed more abundant new bone, mature bone and organized fibrocartilage at the tendon-bone interface at 4 and 8 weeks in groups in which BMP-2 was applied. At 8 weeks, the failure load of groups 1, 2 and 3 was significantly different among the three groups (P = 0.01). After post hoc Tukey test, the failure load of group 2 was significantly higher than that of group 1 (P = 0.01).ConclusionsBMP-2, administrated as described in this study, improved tendon-bone healing and bone formation, resulting in improved biomechanical strength of the tendon-bone junction.     

Stem cell therapies in tendon-bone healing

Tendon-bone insertion injuries such as rotator cuff and anterior cruciate ligament injuries are currently highly common and severe. The key method of treating this kind of injury is the reconstruction operation. The success of this reconstructive process depends on the ability of the graft to incorporate into the bone. Recently, there has been substantial discussion about how to enhance the integration of tendon and bone through biological methods. Stem cells like bone marrow mesenchymal stem cells (MSCs), tendon stem/progenitor cells, synovium-derived MSCs, adipose-derived stem cells, or periosteum-derived periosteal stem cells can self-regenerate and potentially differentiate into different cell types, which have been widely used in tissue repair and regeneration. Thus, we concentrate in this review on the current circumstances of tendon-bone healing using stem cell therapy.     

Vascular endothelial growth factor enhances tendon-bone healing by activating Yes-associated protein for angiogenesis induction and rotator cuff reconstruction in rats

Local angiogenesis following rotator cuff reconstruction is crucial for tendon-bone healing. The current research on the mechanism underlying angiogenesis that promotes tendon-bone healing is scarce. This study investigates the mechanism underlying vascular endothelial growth factor (VEGF)-Hippo signaling pathway's involvement in tendon-bone healing following rotator cuff reconstruction. Verteporfin, the inhibitor of the Yes-associated protein (YAP), was used to mechanically test and analyze two groups of tensile-failure loads following rotator cuff reconstruction and to detect collagen and angiogenesis-related marker expressions in the tendon. The interaction mechanism of the VEGF-Hippo signaling pathway was assessed using human umbilical vein endothelial cells (HUVECs). The diameter of the supraspinatus tendon reduced following verteporfin treatment. Mechanical tests revealed that verteporfin significantly reduces the tensile-failure load of the supraspinatus tendon. Verteporfin significantly reduces collagen 1 (Col 1), Col 3, Angiopoietin 2, CD31, Von Willebrand factor, CTGF, and CYR61 expressions. In HUVECs, VEGF activates VEGF receptors and inhibits LATS and YAP phosphorylation. YAP is then transferred to the nucleus to further activate downstream pathways. Therefore, verteporfin can inhibit VEGF-induced YAP pathway activation by inhibiting YAP activity. Angiogenesis in tendon-bone healing following rotator cuff reconstruction requires VEGF-Hippo signaling pathway synergy.     

Morphological changes of skeletal muscle, tendon and periosteum in the senescence-accelerated mouse (SAMP6): a murine model for senile osteoporosis

SAMP6, a substrain of senescence-accelerated mouse, was developed as an animal model for senile osteoporosis. Previously we observed age-related changes of the bone in SAMP6. In the present study, we investigated the morphology of the skeletal muscle, tendon and periosteum in SAMP6 and age-matched normal mouse SAMR1. We did not find any significant differences between SAMR1 and SAMP6 at 1 and 2 months of age. As compared with SAMR1, the cross-sectional area of type I and type II muscle fibers of the soleus muscle were significantly low in SAMP6 at 8 months of age. The projections in the interface of the muscle-tendon junctions were significantly decreased in SAMP6 at 8 months of age. The number of fibroblasts and the diameter of the tendon collagen fibers in Achilles fiber were significantly reduced in SAMP6 at 8 months of age. The diameter of Sharpey's fiber reduced in SAMP6 at 5 and 8 months of age. Some chondrocytes in the insertions of Achilles tendon and some osteogenic cells in the periosteum showed degenerative changes in SAMP6 at 5 and 8 months of age. The pronounced degenerative changes were detected in the skeletal muscle, muscle-tendon junction, tendon, tendon-bone interface and periosteum in SAMP6 with age. These findings indicated the atrophy of skeletal muscle, degeneration of tendon and periosteum in SAMP6, which may be involved in the bone loss for senile osteoporosis.     

Dynamic multiphoton imaging of acellular dermal matrix scaffolds seeded with mesenchymal stem cells in diabetic wound healing

Significantly effective therapies need to be developed for chronic nonhealing diabetic wounds. In this work, the topical transplantation of mesenchymal stem cell (MSC) seeded on an acellular dermal matrix (ADM) scaffold is proposed as a novel therapeutic strategy for diabetic cutaneous wound healing. GFP‐labeled MSCs were cocultured with an ADM scaffold that was decellularized from normal mouse skin. These cultures were subsequently transplanted as a whole into the full‐thickness cutaneous wound site in streptozotocin‐induced diabetic mice. Wounds treated with MSC‐ADM demonstrated an increased percentage of wound closure. The treatment of MSC‐ADM also greatly increased angiogenesis and rapidly completed the reepithelialization of newly formed skin on diabetic mice. More importantly, multiphoton microscopy was used for the intravital and dynamic monitoring of collagen type I (Col‐I) fibers synthesis via second harmonic generation imaging. The synthesis of Col‐I fibers during diabetic wound healing is of great significance for revealing wound repair mechanisms. In addition, the activity of GFP‐labeled MSCs during wound healing was simultaneously traced via two‐photon excitation fluorescence imaging. Our research offers a novel advanced nonlinear optical imaging method for monitoring the diabetic wound healing process while the ADM and MSCs interact in situ. Schematic of dynamic imaging of ADM scaffolds seeded with mesenchymal stem cells in diabetic wound healing using multiphoton microscopy. PMT, photo‐multiplier tube.      

A CXCL5- and bFGF-dependent effect of PDGF-B-activated fibroblasts in promoting trafficking and differentiation of bone marrow-derived mesenchymal stem cells

Adult bone marrow-derived mesenchymal stem cells (MSCs) are able to differentiate into myofibroblasts and be recruited into wound lesions and contribute to wound healing. The cellular and molecular mechanisms responsible for MSC trafficking and differentiation, however, are poorly understood. Local resting resident fibroblasts are activated after injury and play a critical role in recruiting MSCs. We investigated the role of platelet-derived growth factor-B-activated fibroblasts (PDGF-B-aFBs) in regulating recruitment, migration and differentiation of MSCs from GFP transgenic mice in an in vitro wound healing assay and a novel three-dimensional (3D) model. PDGF-B-aFBs caused significant increases in MSC migration velocity compared to control as demonstrated by time-lapse photography in an in vitro wound healing assay. Consistently, invasion/migration of MSCs into 3D collagen gels was enhanced in the presence of PDGF-B-aFBs. In addition, PDGF-B-aFBs induced differentiation of MSCs into myofibroblast. The regulatory effects of PDGF-B-aFBs are likely to be mediated by basic fibroblast growth factor (bFGF) and epithelial neutrophil activating peptide-78 (ENA-78 or CXCL5) as protein array analysis indicated elevated levels of these two soluble factors in culture supernatant of PDGF-B-aFBs. Blocking antibodies against bFGF and CXCL5 were able to inhibit both trafficking and differentiation of MSCs into 3D collagen gels while supplement of exogenous bFGF and/or CXCL5 promoted invasion/migration of MSCs into 3D collagen gels. Our results reveal that PDGF-B-aFBs play a key role in the recruitment/migration and differentiation of MSCs and implicate a bFGF- and CXCL5-dependent mechanism in mediating these effects.     

The Effect of 58S Bioactive Glass Coating on Polyethylene Terephthalates in Graft-Bone Healing

In this study the effects of surface modification of Polyethylene Terephthalates (PET) fibers with 58S bioactive glasses on osteoblasts proliferation and osseointegration in the tibia-articular tendon-bone healing model were investigated.PET sheets were coated with 58S bioactive glass and uncoated PET sheets were used as a control.Scanning Electron Microscope (SEM) and X-ray photoelectron spectrometer were adopted to analyze the surface characteristics of the fibers.MT3T3-E 1 cells were cultured with the PET fibers and the MTT and ALP were tested at 1,3,5 days.Twenty-four skeletally mature male New Zealand white rabbits were randomly divided into two groups,the 58S-PET group and the PET group.Both groups underwent a surgical procedure to establish a tibia-articular tendon-bone healing model.Mechanical examinations and histological assays were taken to verify the coating effects in vivo.Results of both MTT and ALP tests show significant differences (P ＜ 0.01) between the 58S-PET group and the PET group.At 6 weeks and 12 weeks,the max load-to-failure was significantly higher in the 58S-PET group.In the histological assays,distinct new bone formation was observed only in the 58S-PET group and stronger osseointegration was seen in the 58S-PET group than that in the control group.The 58S-coating on PET could enhance the proliferation and activity of the osteoblasts and therefore promote the new bone formation and tendon-bone healing.     

Mesenchymal stem cell sheet transplantation combined with locally released simvastatin enhances bone formation in a rat tibia osteotomy model

Nonunion of fractured bones is a common clinical problem for orthopedic surgeons. This study aimed to investigate the effects of simvastatin locally applied from calcium sulfate (CS) combined with a mesenchymal stem cell (MSC) sheet on fracture healing. In vitro, the proliferation and differentiation of rat bone marrow–derived MSCs stimulated by simvastatin were investigated. In vivo, an osteotomy model was made in rat tibia, and fractured tibias were treated with CS, CS/simvastatin, CS/MSC sheet or simvastatin-loaded CS with MSC or untreated (control). Tibias were harvested at 2 or 8 weeks and underwent real-time quantitative polymerase chain reaction, x-ray, micro-CT and histological analysis. The expression levels of bone morphogenetic protein 2, alkaline phosphatase, osteocalcin, osteoprotegerin and vascular endothelial growth factor of simvastatin-induced MSCs increased with the concentrations of the simvastatin, significantly higher than those in the MSCs group. At 2 weeks, the CS/simvastatin/MSC sheet group showed significantly higher expressions of bone morphogenetic protein 2, alkaline phosphatase, osteocalcin, osteoprotegerin and vascular endothelial growth factor, with more callus formation around the fracture site compared with the other four groups. At 8 weeks, complete bone union was obtained in the CS/simvastatin/MSC sheet group. By contrast, newly regenerated bone tissue partially bridged the gap in the CS/simvastatin group and the CS/MSC sheet group; the control and CS group showed nonunion of the tibia. These results show that both simvastatin and the MSC sheet contributed to the formation of new bone and that the tibia fracture was completely healed by transplantation of the MSC sheet with locally applied simvastatin. Such MSC sheet with locally applied simvastatin might contribute to the treatment of fractures, bone delayed unions or nonunions in clinical practice.     

Is the tendon embryogenesis process resurrected during tendon healing?

The process of embryonic tendon development, including the nature and purpose of collagen fibril segments, is reviewed. It is proposed that tendon fibrillogenesis of repair is related to the fibrillogenesis of tendon embryonic development. The assembly of collagen fibril segment units into longer fibers occurs on the surface of tendon fibroblasts in embryonic tendon development. The biochemist's view of tendon healing, whereby the spontaneous polymerization of tropocollagen monomers regenerates lost tendon collagen fibers, needs to be reconsidered. Furthermore, the importance of direct fibroblast involvement in collagen fiber reassembly during tendon healing needs to be studied in tendon intrinsic regenerative repair.     

Effect of implanting a soft tissue autograft in a central-third patellar tendon defect: biomechanical and histological comparisons

Previous studies by our laboratory have demonstrated that implanting a stiffer tissue engineered construct at surgery is positively correlated with repair tissue stiffness at 12 weeks. The objective of this study was to test this correlation by implanting a construct that matches normal tissue biomechanical properties. To do this, we utilized a soft tissue patellar tendon autograft to repair a central-third patellar tendon defect. Patellar tendon autograft repairs were contrasted against an unfilled defect repaired by natural healing (NH). We hypothesized that after 12 weeks, patellar tendon autograft repairs would have biomechanical properties superior to NH. Bilateral defects were established in the central-third patellar tendon of skeletally mature (one year old), female New Zealand White rabbits (n?=?10). In one limb, the excised tissue, the patellar tendon autograft, was sutured into the defect site. In the contralateral limb, the defect was left empty (natural healing). After 12 weeks of recovery, the animals were euthanized and their limbs were dedicated to biomechanical (n?=?7) or histological (n?=?3) evaluations. Only stiffness was improved by treatment with patellar tendon autograft relative to natural healing (p?=?0.009). Additionally, neither the patellar tendon autograft nor natural healing repairs regenerated a normal zonal insertion site between the tendon and bone. Immunohistochemical staining for collagen type II demonstrated that fibrocartilage-like tissue was regenerated at the tendon-bone interface for both repairs. However, the tissue was disorganized. Insufficient tissue integration at the tendon-to-bone junction led to repair tissue failure at the insertion site during testing. It is important to re-establish the tendon-to-bone insertion site because it provides joint stability and enables force transmission from muscle to tendon and subsequent loading of the tendon. Without loading, tendon mechanical properties deteriorate. Future studies by our laboratory will investigate potential strategies to improve patellar tendon autograft integration into bone using this model.     

Local administration of allogeneic or autologous bone marrow-derived mesenchymal stromal cells enhances bone formation similarly in distraction osteogenesis

Background aimsDistraction osteogenesis (DO) is a surgical technique to promote bone regeneration that requires a long time for bone healing. Bone marrow-derived mesenchymal stromal cells (MSCs) have been applied to accelerate bone formation in DO. Allogeneic MSCs are attractive, as they could be ready to use in clinics. Whether allogeneic MSCs would have an effect similar to autologous MSCs with regard to promoting bone formation in DO is still unknown. This study compares the effect of autologous MSCs versus allogeneic MSCs on bone formation in a rat DO model.MethodsRat bone marrow-derived MSCs were isolated, characterized and expanded in vitro. Adult rats were subjected to right tibia transverse osteotomy. On the third day of distraction, each rat received one injection of phosphate-buffered saline (PBS), autologous MSCs or allogeneic MSCs at the distraction site. Tibiae were harvested after 28 days of consolidation for micro-computed tomography examination, mechanical test and histological analysis.ResultsResults showed that treatment with both allogeneic and autologous MSCs promoted bone formation, with significantly higher bone mass, mechanical properties and mineral apposition rate as well as expression of angiogenic and bone formation markers at the regeneration sites compared with the PBS-treated group. No statistical difference in bone formation was found between the allogeneic and autologous MSC treatment groups.ConclusionsThis study indicates that allogeneic and autologous MSCs have a similar effect on promoting bone consolidation in DO. MSCs from an allogeneic source could be used off-the-shelf with DO to achieve early bone healing.     

Evaluation of bone marrow derived mesenchymal stem cells for full-thickness wound healing in comparison to tissue engineered chitosan scaffold in rabbit

Background

Chronic wounds present a major challenge in modern medicine. Even under optimal conditions, the healing process may lead to scarring and fibrosis. The ability of mesenchymal stem cells (MSCs) to differentiate into other cell types makes these cells an attractive therapeutic tool for cell transplantation. Both tissue-engineered construct and MSC therapy are among the current wound healing procedures and potential care. Chitosan has been widely applied in tissue engineering because of its biocompatibility and biodegradability.

Adipose tissue‐derived mesenchymal stem cells and keratinocytes co‐culture on gelatin/chitosan/β‐glycerol phosphate nanoscaffold in skin regeneration

Using cell‐based engineered skin is an emerging strategy for treating difficult‐to‐heal wounds. To date, much endeavor has been devoted to the fabrication of appropriate scaffolds with suitable biomechanical properties to support cell viability and growth in the microenvironment of a wound. The aim of this research was to assess the impact of adipose tissue‐derived mesenchymal stem cells (AD‐MSCs) and keratinocytes on gelatin/chitosan/β‐glycerol phosphate (GCGP) nanoscaffold in full‐thickness excisional skin wound healing of rats. For this purpose, AD‐MSCs and keratinocytes were isolated from rats and GCGP nanoscaffolds were electrospun. Through an in vivo study, the percentage of wound closure was assessed on days 7, 14, and 21 after wound induction. Samples were taken from the wound sites in order to evaluate the density of collagen fibers and vessels at 7 and 14 days. Moreover, sampling was done on days 7 and 14 from wound sites to assess the density of collagen fibers and vessels. The wound closure rate was significantly increased in the keratinocytes‐AD‐MSCs‐scaffold (KMS) group compared with other groups. The expressions of vascular endothelial growth factor, collagen type 1, and CD34 were also significantly higher in the KMS group compared with the other groups. These results suggest that the combination of AD‐MSCs and keratinocytes seeded onto GCGP nanoscaffold provides a promising treatment for wound healing.     

Effects of human amniotic membrane grafts combined with marrow mesenchymal stem cells on healing of full-thickness skin defects in rabbits

We have investigated the wound-healing effects of mesenchymal stem cells (MSCs) in combination with human amniotic membrane (HAM) when grafted into full-thickness skin defects of rabbits. Five defects in each of four groups were respectively treated with HAM loaded with autologous MSCs (group A), HAM loaded with allologous MSCs (group B), HAM with injected autologous MSCs (group C), and HAM with injected allologous MSCs (group D). The size of the wounds was calculated for each group at 7, 12, and 15 days after grafting. The wounds were subsequently harvested at 25 days after grafting. Sections stained with hematoxylin and eosin were used to determine the quality of wound healing, as based on the characteristics and amount of granulated tissue in the epidermal and dermal layers. Groups A and B showed the most pronounced effect on wound closure, with statistically significant improvement in wound healing being seen on post-operative days 7, 12, and 15. Although a slight trend toward improved wound healing was seen in group A compared with group B, no statistically significant difference was found at any time point between the two groups. Histological examination of healed wounds from groups A and B showed a thin epidermis with mature differentiation and collagen bundle deposition plus recovered skin appendages in the dermal layer. In contrast, groups C and D showed thickened epidermis with immature epithelial cells and increased fibroblast proliferation with only partially recovered skin appendages in the dermal layer. Thus, the graft of HAM loaded with MSCs played an effective role during the healing of skin defects in rabbits, with no significant difference being observed in wound healing between autologous and allologous MSC transplantation. This study was supported by research funds from Dong-A University.     

Anterior cruciate ligament constructs fabricated from human mesenchymal stem cells in a collagen type I hydrogel

BACKGROUND: Disruptions of the anterior cruciate ligament (ACL) of the knee joint are common and are currently treated using ligament or tendon grafts. In this study, we tested the hypothesis that it is possible to fabricate an ACL construct in vitro using mesenchymal stem cells (MSC) in combination with an optimized collagen type I hydrogel, which is in clinical use for autologous chondrocyte transplantation (ACT). METHODS: ACL constructs were molded using a collagen type I hydrogel containing 5 x 10(5) MSC/mL and non-demineralized bone cylinders at each end of the constructs. The constructs were kept in a horizontal position for 10 days to allow the cells and the gel to remodel and attach to the bone cylinders. Thereafter, cyclic stretching with 1 Hz was performed for 14 days (continuously for 8 h/day) in a specially designed bioreactor. RESULTS: Histochemical analysis for H and E, Masson-Goldner and Azan and immunohistochemical analysis for collagen types I and III, fibronectin and elastin showed elongated fibroblast-like cells embedded in a wavy orientated collagenous tissue, together with a ligament-like extracellular matrix in the cyclic stretched constructs. No orientation of collagen fibers and cells, and no formation of a ligament-like matrix, could be seen in the non-stretched control group cultured in a horizontal position without tension. RT-PCR analysis revealed an increased gene expression of collagen types I and III, fibronectin and elastin in the stretched constructs compared with the non-stretched controls. DISCUSSION: In conclusion, ACL-like constructs from a collagen type I hydrogel, optimized for the reconstruction of ligaments, and MSC have been fabricated. As shown by other investigators, who analyzed the influence of cyclic stretching on the differentiation of MSC, our results indicate a ligament-specific increased protein and gene expression and the formation of a ligament-like extracellular matrix. The fabricated constructs are still too weak for animal experiments or clinical application and current investigations are focusing on the development of a construct with an internal augmentation using biodegradable fibers.     

Diverse effects of type II collagen on osteogenic and adipogenic differentiation of mesenchymal stem cells

Type II collagen is known to modulate chondrogenesis of mesenchymal stem cells (MSCs). In this study, MSCs from human bone marrow aspirates were used to study the modulating effects of type II collagen on MSC differentiation during the early stages of osteogenesis and adipogenesis. With osteogenic induction, MSCs cultured on the type II collagen-coated surface showed an enhanced calcium deposition level with increasing mRNA expressions of RUNX2, osteocalcin, and alkaline phosphatase. A synthetic integrin binding peptide, which specifically interacts with the I-domain of α(1)β(1)/α(2)β(1) integrins significantly blocks the mineralization-enhancing effect of type II collagen. MSCs attached on the type II collagen-coated plates exhibited expanded cell morphology with increasing spreading area, and the pretreatment of cells with integrin α(1)β(1) or α(2)β(1)-blocking antibody reduced the effect. The phosphorylation levels of FAK, ERK, and JNK significantly increased in the MSCs that attached on the type II collagen-coated plates. On the contrary, the mineralization-enhancing effect of type II collagen was diminished by JNK and MEK inhibitors. Furthermore, type II collagen blocked the adipogenic differentiation of MSCs, and this effect is rescued by JNK and MEK inhibitors. In conclusion, type II collagen facilitates osteogenesis and suppresses adipogenesis during early stage MSC differentiation. Such effects are integrin binding-mediated and conducted through FAK-JNK and/or FAK-ERK signaling cascades. These results inspire a novel strategy encompassing type II collagen in bone tissue engineering.     

Anterior Cruciate Ligament Reconstruction in a Rabbit Model Using Silk-Collagen Scaffold and Comparison with Autograft

The objective of the present study was to perform an in vivo assessment of a novel silk-collagen scaffold for anterior cruciate ligament (ACL) reconstruction. First, a silk-collagen scaffold was fabricated by combining sericin-extracted knitted silk fibroin mesh and type I collagen to mimic the components of the ligament. Scaffolds were electron-beam sterilized and rolled up to replace the ACL in 20 rabbits in the scaffold group, and autologous semitendinosus tendons were used to reconstruct the ACL in the autograft control group. At 4 and 16 weeks after surgery, grafts were retrieved and analyzed for neoligament regeneration and tendon-bone healing. To evaluate neoligament regeneration, H&E and immunohistochemical staining was performed, and to assess tendon-bone healing, micro-CT, biomechanical test, H&E and Russell-Movat pentachrome staining were performed. Cell infiltration increased over time in the scaffold group, and abundant fibroblast-like cells were found in the core of the scaffold graft at 16 weeks postoperatively. Tenascin-C was strongly positive in newly regenerated tissue at 4 and 16 weeks postoperatively in the scaffold group, similar to observations in the autograft group. Compared with the autograft group, tendon-bone healing was better in the scaffold group with trabecular bone growth into the scaffold. The results indicate that the silk-collagen scaffold has considerable potential for clinical application.