| 1. Background:Anterior cruciate ligament (ACL) plays a crucial role in the stability and normal kinematics of the knee joint. Due to its limited capacity for regeneration, ACL heals poorly when the ruptured ends are sutured back together. Therefore, the ACL reconstruction is necessary in clinical settings. Now there are three kinds of grafts for the ligament replacements:autografts, allografts and artificial ligaments. The autograft replacements are the preferred choices, especially the 1/3 center of bone-patellar tendon-bone (BPTB) which is the "gold standard" for ACL repair. However, donor site morbidity (e.g., arthritis and patellar tendon rupture) and damage, thus, pain, and the limited supply are the drawbacks of autografts. Allografts eliminate the second site surgery, but they increase the risks of disease transmission, infection and host immunogenic response. Artificial ligaments can be produced in large amount as the suspected size and possess sufficient mechanical properties. While, the synthetic replacements are difficult to be vascularized and integrated with the host bone, and may wear out over time. Currently, the curative effect of the artificial ligament needs to be observed for a long time. The circumstances drive attention to other ideal scaffold with characteristics of wide supply, low immunogenicity and similar biomechanical properties to ACL.Silk protein fibroin is a kind of raw biomaterial. The unique combination of elasticity and strength along with mammalian cell compatibility, slow biodegradability, mild immunogenicity and easy braiding makes silk fibroin an attractive scaffold material for tissue engineering.The braided silk fiber matrix scaffold, the knitted silk-SF sponges and silk-collagen composite scaffold have been broadly studied. Theses scaffolds used to reconstruct ACL in animal model acquired better outcomes. However, these studies only utilized ECM materials to decorate silk fibroin scaffold, not refer to growth factors that have an important effect on modulating cell activities. Currently, the study adopted the mode of promoting vascularization favorable to scaffold to enhance tendon graft--bone healing in anterior cruciate ligament reconstruction is rare.Among utilized ECM materials, SIS is one of the most comprehensive investigated materials including its structure, composition, biological activity and the host constructive remodeling response to it. SIS is mainly composed of collagen I and collagen â…¢.containing less other kinds of collagen, proteoglycan glycosaminoglycan. Most importantly, SIS contains a variety of activated growth factors such as VEGF, BFGF and TGF even have been prepared through a multi-step method. To date, SIS has been extensively used as scaffolds for tissue engineering and regeneration medicine applications. SIS has been verified to induce cell infiltration and vessels formation and angiogenic response favorable to tissue repair on early stage after transplant in vivo. SIS has been extensively used as scaffolds for skin, gullet, cartilage tissue engineering and myocardial infarction regeneration applications.2. ObjectivesIn the progress of tissue regeneration, vessels played an important role in providing oxygen and nutrients and transporting metabolism productions. Therefore, it is vital to tissue regeneration that angiogenesis on early stage of scaffold transplanting in vivo. So the objectives of present study are utilizing SIS that contains a variety of activated growth factors and sufficient collagen I to decorate silk fibroin scaffold and to prepare the small intestinal submucosa (SIS) and silk composite ACL scaffold. Then to evaluate the properties of biomechanics, biocompatibility and the potential of vascularization on early stage after transplant in vivo enhancing the regeneration outcomes.3. Methods3.1 Preparation the small intestinal submucosa (SIS) and small intestinal submucosa-silk composite scaffold.The raw silk was degummed in 0.02 M NaHCO3 solution to remove sericin thoroughly. According to reference, a wire-rope design was chosen to fabricate silk scaffold. By equation model to mirror the rabbit native ACL biomechanics properties, then to fabricate silk scaffold that match native ACL biomechanics properties using computer silk fabrication machine. The braided silk scaffold which was surrounded cylindrically by SIS was the composite scaffold.3.2 The evaluation of the properties of biomechanics and surface ultrastructure.The property of biomechanics was evaluated by biomechanical testing system. Surface ultrastructure of the scaffold materials was evaluated by SEM.3.3 The evaluation of the cytocompatibility of scaffolds.The influence of scaffolds on bone marrow stem cells was evaluated by Live/Dead staining.The effect of extracted scaffold fluids on cell proliferation was evaluated by cell proliferation curve that acquired from the cell counting kit 8 (CCK-8) trial.3.4 The evaluation of the histocompatibility and the potential of vascularization on early stage after heterotopic transplant in vivo.30 Sprague Dawley rats were randomly assigned to 2 groups. The silk scaffold group(S group) and composite scaffold group (SS group) were subcutaneously implanted. After 2 weeks,4 weeks and 8 weeks from the surgery, the specimens were respectively sampled and stained with hematoxylin and eosin (HE) for histological evaluation. The evaluation of histocompatibility was by counting inflammatory reaction cells and potential of vascularization was by counting new vessels.3.5 The influence on reducing or delaying the leakage of synovial fluid into tibia tunnel.20 New Zealand White rabbits were assigned to 2 groups. The silk scaffold group(S group) and composite scaffold group (SS group) and utilized two scaffolds for ACL reconstruction on both knees respectively in rabbit model. Furthermore, a bone window was made on the tibia tunnel. At last, the electric resistance of tendon graft in the bone window was measured and recorded at different time points after 5 ml of 10% NaCl irrigating into the joint and the leaking time was recorded after 5ml of ink solution irrigating into the joint cavity respectively.3.6 The evaluation of surface vascularization of scaffold after ACL reconstruction in rabbit knee.The fresh scaffold implants were acquired at 4 weeks,8 weeks and 12 weeks postoperatively. The sections near the bone tunnel and in the middle were observed by Stereoscopic Microscopes.3.7 The evaluation of remolding of scaffold implants after ACL reconstruction in rabbit knee.Three scaffold implant specimens were acquired at 4 weeks,8 weeks and 12 weeks postoperatively from each group. The specimens were respectively sampled and stained with hematoxylin and eosin (HE) and masson for histological evaluation which focused on the remolding of scaffold implants after ACL reconstruction.3.8 The evaluation of the regenerated ligament-bone junctionThree knee joints from each group were dissected into femur-implant-tibia complexes. The specimens were firstly scanned with micro-CT to evaluate mineralized tissue formation in the intra-tunnel portion of the scaffold implants. Then, the complexes were fixed and decalcified. The ligament-bone insertions were stained with hematoxylin and eosin (HE) for histological evaluation.3.9 Evaluation of biomechanics after ACL reconstruction.Five specimens from each group were used for mechanical test. Each knee joint was dissected of all soft tissue and ligaments except for the implant to create a femur-implant-tibia complex. Finally, evaluate the biomechanics of the complex from load-deformation curve.4. Results4.1 Gross observation of two kinds of scaffoldsThe silk scaffold was braided in the way of a wire-rope using of bundles silk fibroin fibers that was in removal of the contaminating sericin. The processing SIS was elatic collagen film. The composite scaffold consisted of the helical silk bundle inside surrounded by SIS.4.2 The properties of biomechanics and surface ultrastructure.The maximal load of traditional silk scaffold and complex scaffold was respectively 138.62±11.41N and 137.05±16.95N (P>0.05). Besides, the tensile stiffness was respectively 24.65±2.62 N/mm and 24.21±2.39 N/mm (P>0.05).The extention of scaffolds was 9.40%〠10.67% respectively. For both results of maximal loadã€tensile stiffness and extention, there was no significant difference between these two kinds of scaffolds. The maximal load of complex scaffold was matched with rabbit native ACL, while the tensile stiffness significant difference (P<0.05).SEM scans indicated that the superficies of extracted silk fibroin fibers were smoothly and there was rare sericin. The collagen fibres ran straight and approximately parallel to the surface of SIS and there was numbers of porous inside. The composite scaffold consisted of the helical silk bundle inside surrounded by SIS.4.3 The evaluation of cytocompatibility in vitroThe Live/Dead staining showed that the cells had good activity on both scaffolds. However, it was on the composite scaffold that the cells had better extensibility. In addition, the cells on the composite scaffold secreted sufficient ECM.The cell proliferation curve indicated that the OD value was gradually higher along with the culture time and time-depend. There was no significant difference on the OD values from three cell proliferation curves was founded at each time point (p>0.05).4.4 The influence on reducing or delaying the leakage of synovial fluid into tibia tunnel.The time point that electric resistance starting to decrease and the time point that the ink starting to leak were both much later in the SS group than the S group, with statistically differences between the two groups (P<0.05). The time without leakage of ink and sustained time of leakage was much longer in the SS group than the S group, with statistically differences between the two groups (P<0.05).4.5 The evaluation of the histocompatibility and the potential of angiogenesis on early stage after heterotopic transplant in vivo.Our data showed that the inflammatory reaction was present after transplantation. The inflammatory reaction was obviously mild along with time postoperatively. The inflammatory cells in SS group were much less than that in S group at each time point postoperatively and which has a statistically differences (P<0.05).The SIS on the surface of composite scaffold was stared to degrade at 2 weeks postoperatively and there were neovessels in the section. The neovessels were dense and little in lumen.The SIS was largely degraded at 4 weeks postoperatively and there were neovessels inside of composite scaffold. There were numberous vessels in the surface and inside of composite scaffold at 8 weeks postoperatively. The vessel was large in lumen and better mature than before. However, there were no obvious neovessels in silk scaffold at different points postoperatively. The numbers of neovessels were with statistically differences between the two groups (P<0.05).4.6 The surface angiogenesis of scaffold after ACL reconstruction in rabbit knee.The observations from Stereoscopic Microscopes revealed that there was obvious synovium in the surface of composite scaffold implants at 4 weeks postoperatively.The obvious capillarity network of bloodvessels in the surface of synovium at the intraarticular exit of the bone tunnel. The more mature neovessels were extent to the middle of composite scaffold implants at 8 weeks postoperatively. There were large in lumen vessels in the surface of composite scaffold implants. While, there was no obvious neovessels in the surface of silk scaffold implants at 4 weeks postoperatively and only a little synovium covered the surface of silk scaffold at 8 weeks postoperative. The newly-formed synovium was thiner than before and the little bloodvessels were present in the surface of synovium at 12 weeks postoperatively.4.7 The remolding of scaffold implants after ACL reconstruction in rabbit knee.HE staining revealed that:much inflammatory cells infiltrated into silk scaffold implants and no obvious synovium was present in the surface at 4 weeks postoperatively.There was only a little synovium covered the surface of silk fibries scaffold at 8 weeks postoperatively. The silk scaffold implants were tightly combined together and the layer of synovium became thin. While, there were still no obvious neovessels inside the silk scaffold at 12 weeks postoperatively. The composite scaffolds were tightly combined together and much inflammatory cells infiltrated at 4 weeks postoperatively. There were three layers:synovium, the remaining SIS, silk fibrin. There were only two layers and SIS degraded completely at 8 weeks postoperatively. The layer of synovium became thin and much newly-formed neovessels filled inside. The boundaries of the layers in composite scaffold were not be clearly discerned at 12 weeks postoperatively. The implants were fully-filled structure, which was similar to the native ACL.Masson staining revealed that:The silk scaffold implants were mainly composed of numberous remaining silk fabrins and no obvious collagen was observed at 4 weeks postoperatively. The disorganized and loose collagen fabrins started to form in the implants at 8 weeks postoperatively. The collagen fibers were dense but disorganized and loose still at 12 weeks postoperatively. The surface of the composite scaffold was covered with a thin collagen layer and filled with disorganized and loose collagen fabrins at 4 weeks postoperatively. The newly-formed collagen fabrins became dense and organized at 8 weeks postoperatively. The boundaries of the layers in composite scaffold were not clearly discerned and filled with substantial mature collagen fabrins at 12 weeks postoperatively.4.8 assessment of ligament-bone insertion4.81 Histological observationThe fibrovascular connective tissue, progressive collagen fiber-bone anchorage, and fibrocartilage were gradually developed between the implants and bone tunnel in SS group. At 4 weeks postoperatively, only a thin layer of fibrovascular connective tissue was observed on the interface of implant-bone in S group. But in SS group the implant-bone interface was filled with abundant loose fibrovascular granulation tissue with occasional oblique collagen fibers projecting from bone into implants.At 8 weeks, the thin layer of connective tissue became much denser in S group. While in SS group the more organized perpendicular collagen fibers resembling the Sharpey’s fibers were observed to bridge the gap between the implant and bone. After 12 weeks, there was only less dense connective tissue detected on the interface in S group. However, cartilage appeared between the implants and bone in SS group. A gradual transition from bone, cartilage, into the tendon substance was successfully reconstructed.4.82 Micro-CT scanIn both S group and SS group, the consecutive micro-CT images showed that there was no obviously mineralized tissue formed in tibial and femoral bone tunnels at 4 weeks postoperatively. After 12 weeks, there was still no obvious signal detected in bone tunnels of both groups. It indicated that there was less mineralized tissue formation in the intra-tunnel portion of implants in S group and SS group.4.9 Biomechanical analysis postoperativelyFor the failure mode of ligament-bone junction complexes, there was no significant difference between these two kinds of scaffolds.For the maximal tensile load and tensile stiffness, there was no significant difference between these two kinds of scaffolds at 4 weeks postoperatively (p<0.05). While, the SS group showed significantly higher the maximal tensile load and tensile stiffness than S group at 8 and 12 weeks postoperatively (p<0.05).5 ConclusionsThe present study adopted the mode of promoting vascularization to design and construct SIS-silk composite scaffold. The composite ACL scaffold has excellent biomechanical properties, as well as the excellent cytocompatibility and histocompatibility. This composite scaffold has good influence on reducing or delaying the leakage of synovial fluid into tibia tunnel at the early stage reconstruction. Moreover, it has the potential of vascularization on early stage after heterotopic transplant in vivo. The composite scaffold transplants acquired the compatibility of vascularization on early stage of ACL reconstruction in rabbit knee postoperatively. Furthermore, the effect of ligament remodeling was outstanding and a gradual cartilage transition was present in ligament-bone insertion. In conclusion, the short-term effects of SIS-silk composite scaffold on ACL reconstruction were splendid. And it is promising to be an ideal tissue engineering ACL scaffold. |
