The Effects of Extracellular Matrix on Tissue Engineering Construction of Cartilage in Vitro

The effects of various cartilage extracellular matrix on the construction of rabbit growth plate cartilage tissue in vitro were studied. The results show that collagen, proteoglycan and hyaluronic acid can promote the growth of cultured chondrocytes but the effects of various cartilage extracellular matrix(ECM)on chondrocyte differentiation are different. Collagen can promote the hypertrophy of chondrocytes while proteoglycan and hyaluronic acid inhibit the transition of mature chondrocytes into hypertrophied chondrocytes.

Hippocampal Neuron-Derived Extracellular Matrix Coated Nanofibrous Scaffold for Neural Tissue Engineering

The aim of this study is to prepare poly-L-lactide(PLLA)electrospun nanofibrous scaffolds coated with hippocampal neuron-derived extracellular matrix(N-ECM)and construct a novel neural tissue engineering scaffold.Neonatal rat hippocampal neurons were seeded on PLLA nanofibers,and then decellularized to derive a cell-free extracellular matrix loaded N-ECM/PLLA modified scaffolds.The morphology and ingredients of N-ECM/PLLA were observed by scanning electron microscopy(SEM)and immunofluorescence staining respectively,and the cytocompatibility of the composite scaffolds was characterized by cell count kit-8(CCK-8)assay.The N-ECM was clearly identified loading on scaffolds when being imaged via SEM and immunofluorescence staining results showed that the N-ECM was made up of fibronectin and laminin.Most importantly,compared with tissue culture polystyrene and pure scaffolds,N-ECM/PLLA scaffolds could effectively facilitate the proliferation of rat adrenal neuroma cells(PC12 cells),indicating their better cell compatibilities.Based on the combination of N-ECM and PLLA biomaterials,the present study has fabricated a unique and versatile neural tissue engineering scaffold,offering a new thought for future neural tissue engineering.

AAV-mediated expression of p65shRNA and bone morphogenetic protein 4 synergistically enhances chondrocyte regeneration

BACKGROUND:Adeno-associated virus(AAV)gene therapy has been proven to be reliable and safe for the treatment of osteoarthritis in recent years.However,given the complexity of osteoarthritis pathogenesis,single gene manipulation for the treatment of osteoarthritis may not produce satisfactory results.Previous studies have shown that nuclear factorκB could promote the inflammatory pathway in osteoarthritic chondrocytes,and bone morphogenetic protein 4(BMP4)could promote cartilage regeneration.OBJECTIVE:To test whether combined application of AAV-p65shRNA and AAV-BMP4 will yield the synergistic effect on chondrocytes regeneration and osteoarthritis treatment.METHODS:Viral particles containing AAV-p65-shRNA and AAV-BMP4 were prepared.Their efficacy in inhibiting inflammation in chondrocytes and promoting chondrogenesis was assessed in vitro and in vivo by transfecting AAV-p65-shRNA or AAV-BMP4 into cells.The experiments were divided into five groups:PBS group;osteoarthritis group;AAV-BMP4 group;AAV-p65shRNA group;and BMP4-p65shRNA 1:1 group.Samples were collected at 4,12,and 24 weeks postoperatively.Tissue staining,including safranin O and Alcian blue,was applied after collecting articular tissue.Then,the optimal ratio between the two types of transfected viral particles was further investigated to improve the chondrogenic potential of mixed cells in vivo.RESULTS AND CONCLUSION:The combined application of AAV-p65shRNA and AAV-BMP4 together showed a synergistic effect on cartilage regeneration and osteoarthritis treatment.Mixed cells transfected with AAV-p65shRNA and AAV-BMP4 at a 1:1 ratio produced the most extracellular matrix synthesis(P<0.05).In vivo results also revealed that the combination of the two viruses had the highest regenerative potential for osteoarthritic cartilage(P<0.05).In the present study,we also discovered that the combined therapy had the maximum effect when the two viruses were administered in equal proportions.Decreasing either p65shRNA or BMP4 transfected cells resulted in less collagen II synthesis.This implies that inhibiting inflammation by p65shRNA and promoting regeneration by BMP4 are equally important for osteoarthritis treatment.These findings provide a new strategy for the treatment of early osteoarthritis by simultaneously inhibiting cartilage inflammation and promoting cartilage repair.

Decellularized extracellular matrix-based composite scaffolds for tissue engineering and regenerative medicine

Despite the considerable advancements in fabricating polymeric-based scaffolds for tissue engineering,the clinical transformation of these scaffolds remained a big challenge because of the difficulty of simulating native organs/tissues'microenvironment.As a kind of natural tissue-derived biomaterials,decellularized extracellular matrix(dECM)-based scaffolds have gained attention due to their unique biomimetic properties,providing a specific microenvironment suitable for promoting cell proliferation,migration,attachment and regulating differentiation.The medical applications of dECM-based scaffolds have addressed critical challenges,including poor mechanical strength and insufficient stability.For promoting the reconstruction of damaged tissues or organs,dif-ferent types of dECM-based composite platforms have been designed to mimic tissue microenvironment,including by integrating with natural polymer or/and syntenic polymer or adding bioactive factors.In this review,we summarized the research progress of dECM-based composite scaffolds in regenerative medicine,highlighting the critical challenges and future perspectives related to the medical application of these composite materials。

Evaluation of <i>in vivo</i>migration of chondrocytes from tissue-engineered cartilage that was subcutaneously transplanted in mouse model

For regenerative medicine, clarification of in vivo migration of transplanted cells is an important task to secure the safety of transplanted tissue. We had prepared tissue-engineered cartilage consisting of cultured chondrocytes with collagen hydrogel and a biodegradable porous polymer, and we clinically applied it for treatment of craniofacial anomaly. To verify the safety of this tissue-engineered cartilage, we had syngenically transplanted the tissue-engineered cartilage using chondrocytes harvested from EGFP-transgenic mice into subcutaneous pocket of wild type mice, and investigated localizations of transplanted chondrocytes in various organs including cerebrum, lung, liver, spleen, kidney, auricle, gastrocnemius, and femur. After 8 to 24 weeks of the transplantation, accumulation of cartilaginous matrices was observed in tissue-engineered cartilage, while EGFP-positive transplanted chondrocytes were localized in this area. Otherwise, no EGFP was immunohistochemically detected in each organ, suggesting that subcutaneously-transplanted chondrocytes do not migrate to other organs through the circulation. In cartilage tissue engineering using cultured chondrocytes, risk for migration and circulation of transplanted cells seemed negligible, and that ectopic growth of the cells was unlikely to occur, showing that this is safe technique with regard to the in vivo migration of transplanted cells.

Decellularised extracellular matrix-based injectable hydrogels for tissue engineering applications

Decellularised extracellular matrix(dECM)is a biomaterial derived from natural tissues that has attracted considerable attention from tissue engineering researchers due to its exceptional biocompatibility and malleability attributes.These advantageous properties often facilitate natural cell infiltration and tissue reconstruction for regenerative medicine.Due to their excellent fluidity,the injectable hydrogels can be administered in a liquid state and subsequently formed into a gel state in vivo,stabilising the target area and serving in a variety of ways,such as support,repair,and drug release functions.Thus,dECM-based injectable hydrogels have broad prospects for application in complex organ structures and various tissue injury models.This review focuses on exploring research advances in dECM-based injectable hydrogels,primarily focusing on the applications and prospects of dECM hydrogels in tissue engineering.Initially,the recent developments of the dECM-based injectable hydrogels are explained,summarising the different preparation methods with the evaluation of injectable hydrogel properties.Furthermore,some specific examples of the applicability of dECM-based injectable hydrogels are presented.Finally,we summarise the article with interesting prospects and challenges of dECM-based injectable hydrogels,providing insights into the development of these composites in tissue engineering and regenerative medicine.

Engineering an extracellular matrix-functionalized,load-bearing tendon substitute for effective repair of large-to-massive tendon defects

A significant clinical challenge in large-to-massive rotator cuff tendon injuries is the need for sustaining high mechanical demands despite limited tissue regeneration,which often results in clinical repair failure with high retear rates and long-term functional deficiencies.To address this,an innovative tendon substitute named“BioTenoForce”is engineered,which uses(i)tendon extracellular matrix(tECM)’s rich biocomplexity for tendon-specific regeneration and(ii)a mechanically robust,slow degradation polyurethane elastomer to mimic native tendon’s physical attributes for sustaining long-term shoulder movement.Comprehensive assessments revealed outstanding performance of BioTenoForce,characterized by robust core-shell interfacial bonding,human rotator cuff tendon-like mechanical properties,excellent suture retention,biocompatibility,and tendon differentiation of human adipose-derived stem cells.Importantly,BioTenoForce,when used as an interpositional tendon substitute,demonstrated successful integration with regenerative tissue,exhibiting remarkable efficacy in repairing large-to-massive tendon injuries in two animal models.Noteworthy outcomes include durable repair and sustained functionality with no observed breakage/rupture,accelerated recovery of rat gait performance,and>1 cm rabbit tendon regeneration with native tendon-like biomechanical attributes.The regenerated tissues showed tendon-like,wavy,aligned matrix structure,which starkly contrasts with the typical disorganized scar tissue observed after tendon injury,and was strongly correlated with tissue stiffness.Our simple yet versatile approach offers a dual-pronged,broadly applicable strategy that overcomes the limitations of poor regeneration and stringent biomechanical requirements,particularly essential for substantial defects in tendon and other load-bearing tissues.

Biocompatibility Evaluation of Vessel Extracellular Matrix as a Matrix for Urethral Reconstruction

The objective of this study was to evaluate the biocompatibility of vessel extracellular matrix （VECM） from rabbit and to discuss the feasibility of vessel extracellular matrix as a matrix for urethral reconstruction. Primary cultured bladder smooth muscle cells isolated from New Zealand rabbits were implanted on VECM .The effects of VECM on rabbit bladder smooth muscle cells （RBSMCs） metabolic activity, attachment, proliferation were monitored in vitro with the aid of an inverted light microscope and a scanning electron microscope. The cell viability was monitored by MTT（methythiazolye tetrazolium bromide） after 1, 3, 5 days seeding. The in vivo tissue response to VECM was investigated by implanting them into the subcutaneous of rabbits. VECM exhibited a nontoxic and bioactive effect on RBSMCs. RBSMCs could be attached to and proliferated on VECM and maintained their morphologies. MTT assay showed RBSMCs cultured with the extracts of VECM were not significantly different from those of negative controls. In vivo, VECM demonstrated a favorable tissue compatibility without tissue necrosis, fibrosis and other abnormal response. VECM exhibited nontoxic and bioactive effects on RBSMC. It is a suitable material for urethral reconstruction.

Nanofibrous Scaffold Containing Osteoblast-Derived Extracellular Matrix for the Proliferation of Bone Marrow Mesenchymal Stem Cells

Extracellular matrix( ECM) plays a prominent role in establishing and maintaining an appropriate microenvironment for tissue regeneration. The aims of this study were to construct a tissue engineered scaffold by reconstituting osteoblast cell-derived ECM( O-ECM) on the electrospun nanofibrous scaffold,and further to evaluate its subsequent application for promoting the proliferation of bone marrow mesenchymal stem cells( BMSCs). To engineer a biomimetic scaffold, calvarial osteoblasts and electrospun poly-llactic acid( PLLA) nanofibers were prepared and subjected to decellularize for O-ECM deposition. To evaluate and characterize the O-ECM/PLLA scaffold, the morphology was examined and several specific mark proteins of osteoblasts matrix were evaluated.Furthermore,the cell counting kit-8( CCK-8) assay was used to detect the proliferation of the BMSCs cultivated on the O-ECM/PLLA scaffold. The results indicated O-ECM/PLLA scaffold was loaded with Collagen I, Fibronectin, and Laminin, as the composition of the marrow ECM. After decellularization,O-ECM deposition was observed in O-ECM/PLLA scaffold. Moreover,the O-ECM/PLLA scaffold could significantly enhance the proliferation of BMSCs,suggesting better cytocompatibility compared to the other groups tested. Taken together,a biomimetic scaffold based on the joint use of O-ECM and PLLA biomaterials,which represents a promising approach to bone tissue engineering, facilitates the expansion of BMSCs in vitro.

GW842166X Alleviates Osteoarthritis by Repressing LPS-mediated Chondrocyte Catabolism in Mice

Objective To explore the role and underlying mechanism of GW842166X on osteoarthritis and osteoarthritis-associated abnormal catabolism.Methods The extracted mouse chondrocytes were treated with GW842166X followed by lipopolysaccharide(LPS).The chondrocytes were divided into the control group,LPS group,LPS+50 nmol/L GW842166X group,and LPS+100 nmol/L GW842166X group.The cytotoxicity of GW842166X was tested using the CCK-8 assay.Western blot,RT-qPCR,and ELISA were applied to evaluate the expression of the inflammatory biomarkers in mouse chondrocytes.The expression of extracellular matrix molecules was detected by the Western blot,RT-qPCR,and immunofluorescence.Additionally,the activity of NF-κB was checked by the Western blot and immunofluorescence.The mouse Hulth models were generated to examine the in vivo effects of GW842166X on osteoarthritis.Hematoxylin and eosin staining,safranin O/fast green staining,and immunohistochemistry were applied to detect the histological changes.Results GW842166X below 200µmol/L had no cytotoxicity on the mouse chondrocytes.LPS-induced high expression of TGF-β1,IL-10,TNF-α,and IL-6 was significantly reduced by GW842166X.In addition,GW842166X upregulated the expression of aggrecan and collagen type III,which was downregulated after the LPS stimulation.The upregulated expression of ADAMTS-5 and MMP-13 by LPS stimulation was dropped in response to the GW842166X treatment.Furthermore,LPS decreased the IκBαexpression in the cytoplasm and increased the nuclear p65 expression.However,these changes were reversed by the GW842166X pretreatment.Moreover,the damages in the knees caused by the Hulth surgery in mice were restored by GW842166X.Conclusion GW842166X impeded the LPS-mediated catabolism in mouse chondrocytes,thereby inhibiting the progression of osteoarthritis.

Decellularized extracellular matrix particle-based biomaterials for cartilage repair applications

Cartilage Decellularized ExtraCellular Matrix(dECM)materials have shown promising cartilage regenera-tion capacity due to their chondrogenic bioactivity.However,the limited retention of ECM components and the reduced integrity of functional ECM molecules during traditional decellularization processes im-pair the biomimicry of these materials.The current study aims to fabricate biomimetic materials con-taining decellularized cartilage particles that have an intact molecular structure and native composition as biomaterial inks and hydrogels for cartilage repair.For this,we established a novel two-fraction de-cellularization strategy for the preparation of reconstituted dECM(rdECM)particles by mixing the two-fraction components,as well as a one-fraction decellularization strategy for the preparation of biomimetic dECM(bdECM)particles.Hyaluronic acid-tyramine(THA)hydrogels containing rdECM or bdECM particles were produced and characterized via rheological test,swelling and stability evaluation,and compression test.The results showed that our novel decellularization strategies preserved intact proteoglycans and collagen at a higher retention rate with adequate DNA removal compared to traditional methods of de-cellularization.The addition of rdECM or bdECM particles significantly increased the shear moduli of the THA bioinks while preserving their shear-thinning properties.bdECM particle-embedded THA hydrogels also achieved long-term stability with a swelling ratio of 70%and high retention of glycosaminoglycans and collagen after long-term incubation,while rdECM particle-embedded THA hydrogels showed unsat-isfactory stability as self-standing biomaterials.Compared to pure THA hydrogels,the addition of bdECM particles significantly enhanced the compression moduli.In summary,our decellularization methods are successful in the retention of functional and intact cartilage components with high yield.Both rdECM and bdECM particles can be supplemented in THA bioinks for biomimetic cartilage 3D printing.Hydro-gels with cartilage bdECM particles possess the functional structure and the natural composition of car-tilage ECM,long-term stability,and enhanced mechanical properties,and are promising biomaterials for cartilage repair.

Growth factor delivery using extracellular matrix-mimicking substrates for musculoskeletal tissue engineering and repair

Therapeutic approaches for musculoskeletal tissue regeneration commonly employ growth factors(GFs)to influence neighboring cells and promote migration,proliferation,or differentiation.Despite promising results in preclinical models,the use of inductive biomacromolecules has achieved limited success in translation to the clinic.The field has yet to sufficiently overcome substantial hurdles such as poor spatiotemporal control and supraphysiological dosages,which commonly result in detrimental side effects.Physiological presentation and retention of biomacromolecules is regulated by the extracellular matrix(ECM),which acts as a reservoir for GFs via electrostatic interactions.Advances in the manipulation of extracellular proteins,decellularized tissues,and synthetic ECM-mimetic applications across a range of biomaterials have increased the ability to direct the presentation of GFs.Successful application of biomaterial technologies utilizing ECM mimetics increases tissue regeneration without the reliance on supraphysiological doses of inductive biomacromolecules.This review describes recent strategies to manage GF presentation using ECM-mimetic substrates for the regeneration of bone,cartilage,and muscle.

In vitro cartilage production using an extracellular matrix-derived scaffold and bone marrow-derived mesenchymal stem cells

Background Cartilage repair is a challenging research area because of the limited healing capacity of adult articular cartilage.We had previously developed a natural,human cartilage extracellular matrix （ECM）-derived scaffold for in vivo cartilage tissue engineering in nude mice.However,before these scaffolds can be used in clinical applications in vivo,the in vitro effects should be further explored.Methods We produced cartilage in vitro using a natural cartilage ECM-derived scaffold.The scaffolds were fabricated by combining a decellularization procedure with a freeze-drying technique and were characterized by scanning electron microscopy （SEM）,micro-computed tomography （micro-CT）,histological staining,cytotoxicity assay,biochemical and biomechanical analysis.After being chondrogenically induced,the induction results of BMSCs were analyzed by histology and Immunohisto-chemistry.The attachment and viability assessment of the cells on scaffolds were analyzed using SEM and LIVE/DEAD staining.Cell-scaffold constructs cultured in vitro for 1 week and 3 weeks were analyzed using histological and immunohistochemical methods.Results SEM and micro-CT revealed a 3-D interconnected porous structure.The majority of the cartilage ECM was found in the scaffold following the removal of cellular debris,and stained positive for safranin O and collagen Ⅱ.Viability staining indicated no cytotoxic effects of the scaffold.Biochemical analysis showed that collagen content was （708.2±44.7）μg/mg,with GAG （254.7±25.9） μg/mg.Mechanical testing showed the compression moduli （E） were （1.226±0.288） and （0.052±0.007） MPa in dry and wet conditions,respectively.Isolated canine bone marrow-derived stem cells （BMSCs） were induced down a chondrogenic pathway,labeled with PKH26,and seeded onto the scaffold.Immunofluorescent staining of the cell-scaffold constructs indicated that chondrocyte-like cells were derived from seeded BMSCs and excreted ECM.The cell-scaffold constructs contained pink,smooth and translucent cartilage-like tissue after 3 weeks of culture.We observed evenly distributed cartilage ECM proteoglycans and collagen type Ⅱ around seeded BMSCs on the surface and inside the pores throughout the scaffold.Conclusion This study stuggests that a cartilage ECM scaffold holds much promise for in vitro cartilage tissue engineering.

Cardiac tissue-derived extracellular matrix scaffolds for myocardial repair:advantages and challenges

Decellularized extracellular matrix(dECM)derived from myocardium has been widely explored as a nature scaffold for cardiac tissue engineering applications.Cardiac dECM offers many unique advantages such as preservation of organ-specific ECM microstructure and composition,demonstration of tissue-mimetic mechanical properties and retention of biochemical cues in favor of subsequent recellularization.However,current processes of dECM decellularization and recellularization still face many challenges including the need for balance between cell removal and extracellular matrix preservation,efficient recellularization of dECM for obtaining homogenous cell distribution,tailoring material properties of dECM for enhancing bioactivity and prevascularization of thick dECM.This review summarizes the recent progresses of using dECM scaffold for cardiac repair and discusses its major advantages and challenges for producing biomimetic cardiac patch.

Evaluation of an extracellular matrix-derived acellular biphasic scaffold/cell construct in the repair of a large articular high-load-bearing osteochondral defect in a canine model

Osteochondral lesion repair is a challenging area of orthopedic surgery. Here we aimed to develop an extracellular matrix-derived, integrated, biphasic scaffold and to investigate the regeneration potential of the scaffold loaded with chondrogenically-induced bone marrow-derived mesenchymal stem cells （BMSCs） in the repair of a large, high-load-bearing, osteochondral defect in a canine model. Methods The biphasic scaffolds were fabricated by combining a decellularization procedure with a freeze-drying technique and characterized by scanning electron microscopy （SEM） and micro-computed tomography （micro-CT）. Osteochondral constructs were fabricated in vitro using chondrogenically-induced BMSCs and a biphasic scaffold, then assessed by SEM for cell attachment. Osteochondral defects （4.2 mm （diameter） ×6 mm （depth）） were created in canine femoral condyles and treated with a construct of the biphasic scaffold/chondrogenically-induced BMSCs or with a cell-free scaffold （control group）. The repaired defects were evaluated for gross morphology and by histological, biochemical, biomechanical and micro-CT analyses at 3 and 6 months post-implantation. Results The osteochondral defects of the experimental group showed better repair than those of the control group. Statistical analysis demonstrated that the macroscopic and histologic grading scores of the experimental group were always higher than those of the control group, and that the scores for the experimental group at 6 months were significantly higher than those at 3 months. The cartilage stiffness in the experimental group （6 months） was （6.95±0.79） N/mm, 70.77% of normal cartilage; osteochondral bone stiffness in the experimental group was （158.16±24.30） N/mm, 74.95% of normal tissue; glycosaminoglycan content of tissue-engineered neocartilage was （218±21.6） tJg/mg （dry weight）, 84.82% of native cartilage. Micro-CT analysis of the subchondral bone showed mature trabecular bone regularly formed at 3 and 6 months, with no significant difference between the experimental and control groups. Conclusion The extracellular matrix-derived, integrated, biphasic scaffold shows potential for the repair of large, high-load-bearing osteochondral defects.

Reconstruction of rabbit urethra using urethral extracellular matrix

Background Urethral reconstruction for both congenital and acquired etiologies remains a challenge for most urologic surgeons Tissue engineering has been proposed as a strategy for urethral reconstruction The purpose of this study was to determine whether a naturally derived extracellular matrix substitute developed for urethral reconstruction would be suitable for urethral repair in an animal model Methods A urethral segmental defect was created in 20 male rabbits The urethral extracellular matrix, obtained and processed from rabbit urethral tissue, was trimmed and transplanted to repair the urethral defect Then, the regenerated segment was studied histologically by haematoxylin eosin staining and Van Gieson staining at 10 days, 3 weeks, 6 weeks, and 24 weeks postoperation Retrograde urethrography was used to evaluate the function of the regenerated urethras of 4 rabbits 10 and 24 weeks after the operation The urodynamics of 4 rabbits from the experimental group and control group Ⅰ were assessed and compared In addition, 4 experimental group rabbits were examined by a urethroscope 24 weeks after the operation Results At 10 days after operation, epithelial cells had migrated from each side, and small vessels were observed in the extracellular matrix The matrix and adjacent areas of the host tissue were infiltrated with inflammatory cells The epithelium covered the extracellular matrix fully at 3 weeks postoperation Well formed smooth muscle cells were first confirmed after 6 weeks, at which point the inflammatory cells had disappeared At 24 weeks postoperation, the regenerated tissue was equivalent to the normal urethra Urethrography and urodynamic evaluations showed that there was no difference between normal tissue and regenerated tissue Conclusions Urethral extracellular matrix appears to be a useful material for urethral repair in rabbits The matrix can be processed easily and has good characteristics for tissue handling and urethral function

Crosslinking strategies for preparation of extracellular matrix-derived cardiovascular scaffolds

Heart valve and blood vessel replacement using artificial prostheses is an effective strategy for the treatment of cardiovascular disease at terminal stage.Natural extracellular matrix(ECM)-derived materials(decellularized allogeneic or xenogenic tissues)have received extensive attention as the cardiovascular scaffold.However,the bioprosthetic grafts usually far less durable and undergo calcification and progressive structural deterioration.Glutaraldehyde(GA)is a commonly used crosslinking agent for improving biocompatibility and durability of the natural scaffold materials.However,the nature ECM and GA-crosslinked materials may result in calcification and eventually lead to the transplant failure.Therefore,studies have been conducted to explore new crosslinking agents.In this review,we mainly focused on research progress of ECM-derived cardiovascular scaffolds and their crosslinking strategies.

Peripheral nerve allograft prepared by an acellular,hypotonic tissue engineering method

BACKGROUND：Tissue engineered acellular nerves are good autologous nerve substitutes. Acellular peripheral nerves prepared using a conventional chemical extraction method cause a great deal of damage to nerve structures, and the allograff affects the nerve regeneration following transplantation.OBJECTIVE：To prepare peripheral nerve grafts through an acellular tissue engineering method, and observe their histology, ultrastructure, protein components and histocompatibility.DESIGN, TIME AND SETTING：A randomized, controlled, in vivo nerve tissue engineering experiment was performed at the Department of Biochemistry and Molecular Biology, Shenyang Medical College, China, from September 2006 to June 2007.MATERIALS：Triton X-100, Pepstatin A, Aprotinin and Leupeptin were purchased from Sigma, USA; Tris （hydroxymethyl） aminomethane was purchased from Gibco, USA.METHODS：The bilateral sciatic nerves of Wistar rats were harvested, treated with 0.05 mol/L Tris-HCI buffer, followed by proteinase inhibitor and Triton X-100 to prepare acellular peripheral nerves. The nerves were implanted in the quadriceps femoris muscle of healthy Wistar rats.MAIN OUTCOME MEASURES：Tissue structure and ultrastructure of acellular peripheral nerves were observed by optical microscopy and scanning electron microscopy. Growth associated proteins were analyzed by sodium dodecyl sulfate polyacrylamide gel electrophoresis. Nerve allograft and the surrounding muscles were observed by hematoxylin-eosin staining.RESULTS：Acellular treatment eliminated Schwann cells, epineurium or perineurium cells, myelin sheaths and axons of nerve fibers in normal peripheral nerves, while the spatial structure, comprising basement membrane tubes of Schwann cells and the extracellular matrix of perineurium and nerve fascicles was maintained. Protein bands at the region of 30 kD were no longer visible, had slightly decreased at 43 kD and remained unchanged at 65 kD. Following implantation for 7 days, epineurium cells were absorbed. However, increased fibroblasts, decreased newly-generated capillaries and maturation of granulation tissue were observed.CONCLUSION：The acellular nerve allograft prepared through the use of a hypotonic, acellular method displays good histocompatibility, eliminates immune substances and retains growth associated proteins that induce the growth of the neural axis. In addition, this method provides an ideal scaffold to construct artificial nerves.

A Novel Biomaterial for Cartilage Repair Generated by Self-Assembly: Creation of a Self-Organized Articular Cartilage-Like Tissue

Recently, attention has been drawn to tissue engineering and other novel techniques aimed at reconstruction of the joint. Regarding articular cartilage tissue engineering, three-dimensional materials created in vitro by cultivation of autologous chondrocytes or mesenchymal stem cells with a collagen gel have been implanted to replace defective parts of the articular cartilage in limited cases with the diseases such as trauma or arthritis. However, several passages of chondrocyte culture are required to obtain a sufficient number of cells for tissue engineering. Additionally, several other problems arise including dedifferentiation of chondrocytes during cell culture, which need to be solved from a viewpoint of cellular resources. The purpose of our study is to create a novel biomaterial possessing functions and structures comparable to native hyaline articular cartilage by utilizing the physicochemical properties of the cartilage matrix components themselves, in other words, employing a self-assembly technique instead of using chondrocytes to produce cartilage matrices eventually leading to articular cartilage tissue formation. We verified the conditions and accuracy of the self-organization process and analyzed the resulting micro structure using electron beam microscopy in order to study the technique involved in the self-organization which would be applicable to creation of cartilage-like tissue. We demonstrated that self-assembly of several cartilage components including type II collagen, proteoglycan and hyaluronic acid could construct self-assembled cartilage-like tissues characterized by nano composite structures comparable to human articular cartilage and by low friction coefficients as small as those of native cartilage.