Patient-derived extracellular matrix demonstrates role of COL3A1 in blood vessel mechanics.

Vascular Ehlers-Danlos Syndrome (vEDS) is a rare autosomal dominant disease caused by mutations in the COL3A1 gene, which renders patients susceptible to aneurysm and arterial dissection and rupture. To determine the role of COL3A1 variants in the biochemical and biophysical properties of human arterial ECM, we developed a method for synthesizing ECM directly from vEDS donor fibroblasts. We found that the protein content of the ECM generated from vEDS donor fibroblasts differed significantly from ECM from healthy donors, including upregulation of collagen subtypes and other proteins related to ECM structural integrity. We further found that ECM generated from a donor with a glycine substitution mutation was characterized by increased glycosaminoglycan content and unique viscoelastic mechanical properties, including increased time constant for stress relaxation, resulting in a decrease in migratory speed of human aortic endothelial cells when seeded on the ECM. Collectively, these results demonstrate that vEDS patient-derived fibroblasts harboring COL3A1 mutations synthesize ECM that differs in composition, structure, and mechanical properties from healthy donors. These results further suggest that ECM mechanical properties could serve as a prognostic indicator for patients with vEDS, and the insights provided by the approach demonstrate the broader utility of cell-derived ECM in disease modeling. STATEMENT OF SIGNIFICANCE: The role of collagen III ECM mechanics remains unclear, despite reported roles in diseases including fibrosis and cancer. Here, we generate fibrous, collagen-rich ECM from primary donor cells from patients with vascular Ehlers-Danlos syndrome (vEDS), a disease caused by mutations in the gene that encodes collagen III. We observe that ECM grown from vEDS patients is characterized by unique mechanical signatures, including altered viscoelastic properties. By quantifying the structural, biochemical, and mechanical properties of patient-derived ECM, we identify potential drug targets for vEDS, while defining a role for collagen III in ECM mechanics more broadly. Furthermore, the structure/function relationships of collagen III in ECM assembly and mechanics will inform the design of substrates for tissue engineering and regenerative medicine.

Mesenchymal Stromal Cells Derived Conditioned Medium in Pulmonary Fibrosis: A Systematic Review and Meta-analysis.

BACKGROUND: A definitive conclusion on the efficacy of mesenchymal stromal cells-derived conditioned medium (MSCs-CM) in pulmonary fibrosis has not yet been reached. Therefore, the present meta-analysis intends to investigate the efficacy of MSCs-CM administration on improvement of pulmonary fibrosis. METHODS: An extensive search was performed on the Medline, Embase, Scopus and Web of Science databases by the end of August 2019. Outcomes in the present study included pulmonary fibrosis score, lung collagen deposition, lung collagen expression, transforming growth factor ß1 (TGF-ß1) expression and interleukin-6 expression. Finally, the data were pooled and an overall standardized mean difference (SMD) with a 95% confidence interval (CI) was reported. RESULTS: Data from seven studies were included. Analyses showed that administration of MSCs-CM significantly improved pulmonary fibrosis (SMD = -2.36; 95% CI: -3.21, -1.51). MSCs-CM administration also attenuated lung collagen deposition (SMD = -1.70; 95% CI: -2.18, -1.23) and decreased expression of type I collagen (SMD = -6.27; 95% CI: -11.00, -1.55), type III collagen (SMD = -5.16; 95% CI: -9.86, -0.47), TGF- ß1 (SMD = -3.36; 95% CI: - 5.62, -1.09) and interleukin-6 (SMD = -1.69; 95% CI: - 3.14, -0.24). CONCLUSION: The present meta-analysis showed that administration of MSCs-CM improves pulmonary fibrosis. It seems that the effect of MSCs-CM was mediated by reducing collagen deposition as well as inhibiting the production of inflammatory chemokines such as TGF-ß1 and interleukin 6 (IL-6). Since there is no evidence on the efficacy of MSCs-CM in large animals, further studies are needed to translate the finding to clinical studies.

Using synthetic peptides and recombinant collagen to understand DDR-collagen interactions.

The discoidin domain receptors, DDR1 and DDR2, are a subfamily of receptor tyrosine kinases that are activated upon binding to collagen. DDR-collagen interactions play an important role in cell proliferation and migration. Over the past few decades, synthetic peptides and recombinant collagen have been developed as tools to study the biophysical characteristics of collagen and various protein-collagen interactions. Herein we review how these techniques have been used to understand DDR-collagen interactions. Using synthetic collagen-like peptides, the GVM-GFO motif has been found to be the major binding site on collagens II and III for DDR1 and DDR2. An X-ray co-crystal structure of the DDR2 DS domain bound to a synthetic collagen-like peptide containing the GVM-GFO motif further provides molecular details of the DDR-collagen interactions. Recombinant collagen has also been used to provide further validation of the GVM-GFO binding motif. Although GVM-GFO has been defined as the minimal binding site, in synthetic peptide studies at least two triplets N-terminal to the essential GVM-GFO binding motif in collagen III sequence are needed for DDR2 activation at high peptide concentrations.

Characterization by high-resolution crystal structure analysis of a triple-helix region of human collagen type III with potent cell adhesion activity.

Collagen is one of the most abundant and important proteins in the human body. Human collagen type III (hCOL3A1) belongs to the fibril-forming collagens and is widely distributed in extensible connective tissue like skin, internal organs, or the vascular system. It plays key roles in wound healing, collagen fibrillogenesis, and normal cardiovascular development in human. The charged residues are considered to be an important characteristic of hCOL3A1, especially for collagen binding and recognition. Here we found that a triple helix fragment of hCOL3A1, Gly489-Gly510, contained multiple charged residues, as well as representative Glu-Lys-Gly and Glu-Arg-Gly charged triplets. We solved the crystal structure of this new fragment to a high-resolution of 1.50 Šand identified some important conformations of this new triple-helix region, including strong hydrogen bonds in interchain and interhelical interactions in addition to obvious flexible bending for the triple helix. We also found that the synthetic collagen peptides around this region exhibited potent activities through integrin-mediated peptide-membrane interaction. We then developed a method to produce a recombinant protein consisting of 16 tandem repeats of the triple-helix fragment of hCOL3A1 with strong activity without cytotoxicity. These results provide a strong base for further functional studies of human collagen type III and the method developed in this study can be applied to produce hCOL3A1-derived proteins or other tandem-repeat proteins with membrane adhesion activity.

Wet spinning and riboflavin crosslinking of collagen type I/III filaments.

Reconstituted fibrillary collagen is one of the most advantageous biomaterials for biomedical applications. The objective of the research project described in this paper was to evaluate whether riboflavin-induced photo-crosslinking could be used as a non-toxic alternative to glutaraldehyde (GA)-crosslinking for the preparation of wet spun collagen filaments. Collagen filaments were produced on a laboratory wet spinning line and crosslinked with GA or riboflavin with and without UV exposure. Based on mechanical and thermal analyses, it was concluded that the combination of riboflavin and UV light leads to crosslinked collagen filaments having improved mechanical and thermal properties. Furthermore, riboflavin-crosslinked filaments exhibited a higher cytocompatibility for human mesenchymal stem cells compared to GA-crosslinked filaments.

Functional disorders of the temporomandibular joints: Internal derangement of the temporomandibular joint.

Temporomandibular joint (TMJ) is one of the most complex joints of the human body. Due to its unique movement, in terms of combination of rotation and translator movement, disc of the joint plays an important role to maintain its normal function. In order to sustain the normal function of the TMJ, disc must be kept in proper position as well as maintain normal shape in all circumstances. Once the disc is not any more in its normal position during function of the joint, disturbance of the joint can be occurred which will lead to subsequent distortion of the disc. Shape of the disc can be influenced by many factors i.e.: abnormal function or composition of the disc itself. Etiology of the internal derangement of the disc remains controversial. Multifactorial theory has been postulated in most of previous manuscripts. Disc is composed of mainly extracellular matrix. Abnormal proportion of collagen type I & III may also leads to joint hypermobility which may be also a predisposing factor of this disorder. Thus it can be recognized as local manifestation of a systemic disorder. Different treatment modalities with from conservative treatment to surgical intervention distinct success rate have been reported. Recently treatment with extracellular matrix injection becomes more and more popular to strengthen the joint itself. Since multifactorial in character, the best solution of the treatment modalities should be aimed to resolve possible etiology from different aspects. Team work may be indication to reach satisfied results.

Autologous Adipose-Derived Tissue Matrix Part I: Biologic Characteristics.

BACKGROUND: Autologous collagen is an ideal soft tissue filler and may serve as a matrix for stem cell implantation and growth. Procurement of autologous collagen has been limited, though, secondary to a sufficient source. Liposuction is a widely performed and could be a source of autologous collagen. OBJECTIVES: The amount of collagen and its composition in liposuctioned fat remains unknown. The purpose of this research was to characterize an adipose-derived tissue-based product created using ultrasonic cavitation and cryo-grinding. This study evaluated the cellular and protein composition of the final product. METHODS: Fat was obtained from individuals undergoing routine liposuction and was processed by a 2 step process to obtain only the connective tissue. The tissue was then evaluated by scanning electronic microscope, Western blot analysis, and flow cytometry. RESULTS: Liposuctioned fat was obtained from 10 individuals with an average of 298 mL per subject. After processing an average of 1 mL of collagen matrix was obtained from each 100 mL of fat. Significant viable cell markers were present in descending order for adipocytes > CD90+ > CD105+ > CD45+ > CD19+ > CD144+ > CD34+. Western blot analysis showed collagen type II, III, IV, and other proteins. Scanning electronic microscope study showed a regular pattern of cross-linked, helical collagen. Additionally, vital staing demonstrated that the cells were still viable after processing. CONCLUSIONS: Collagen and cells can be easily obtained from liposuctioned fat by ultrasonic separation without alteration of the overall cellular composition of the tissue. Implantation results in new collagen and cellular growth. Collagen matrix with viable cells for autologous use can be obtained from liposuctioned fat and may provide long term results. LEVEL OF EVIDENCE: 5.

Autologous Adipose-Derived Tissue Matrix Part II: Implantation Biology.

BACKGROUND: In part 1 of this study it was shown that liposuctioned fat could be a sufficient source of autologous collagen for use as a filler or in reconstruction. The collagen composition in liposuctioned fat was shown to form a cross-linked helical matrix composed of types II, III, and IV. Additionally, viable adipocytes and fibroblasts among other cells were found. OBJECTIVES: The purpose of this research was to study the biology of this matrix after subsequent implantation compared to Juvederm (Allergan, Parsippany, NJ) common soft tissue filler. METHODS: Fat was obtained from individuals undergoing routine liposuction and was processed by a two-step process to obtain a connective tissue matrix. The matrix was then cryo-frozen for a minimum of 4 weeks after which it was thawed and implanted in 46 nude mice. Juvederm Ultra was used as the control article and the animals followed for one year. RESULTS: Liposuctioned fat was obtained from 10 individuals and processed as previously described. Mice were harvested at 3, 6, 9, and 12 months and histology obtained. There were no adverse effects from either article and the bio-reactivity rating was 0. The implanted collagen compared favorably to Juvederm at all stages and was found to be replaced by new collagen and fat. CONCLUSIONS: A collagen matrix with viable cells for autologous use can be obtained from liposuctioned fat which has been processed and cryo-frozen. The material lasts at least one year and is slowly replaced by new collagenand fat. LEVEL OF EVIDENCE: 5.

Identification of a missense mutation of COL3A1 in a Chinese family with atypical Ehlers-Danlos syndrome using targeted next-generation sequencing.

Aortopathy represents an important cause of mortality in industrialized countries, with a number of genes identified as predispose factors. It can be difficult to identify the genetic lesions underlying this disorder, particularly when the phenotype is atypical. The present study performed targeted next­generation sequencing of 428 genes associated with cardiovascular diseases in a family with aortopathy, the proband of which presented with abdominal aortic aneurysm rupture only, with tissue fragility noted in surgery. After targeted capture, sequencing and bioinformatics analysis, a missense mutation, p.A1259T, was identified in the collagen type III α1 (COL3A1) gene and co­segregated with the disease in the family. Crystal structure modeling revealed abnormal hydrogen bonds generated by the mutation, which likely affected the spatial structure of the procollagen C­propeptide. Mutations in the procollagen C­propeptide are rare and genotype­phenotype correlation may explain the atypical manifestations of affected individuals. The results of the present study suggested that targeted gene capture combined with next­generation sequencing can serve as a useful technique in the genetic diagnosis of aortopathy, particularly in the content of an atypical phenotype.

Characterization of the ultrashort-TE (UTE) MR collagen signal.

Although current cardiovascular MR (CMR) techniques for the detection of myocardial fibrosis have shown promise, they nevertheless depend on gadolinium-based contrast agents and are not specific to collagen. In particular, the diagnosis of diffuse myocardial fibrosis, a precursor of heart failure, would benefit from a non-invasive imaging technique that can detect collagen directly. Such a method could potentially replace the need for endomyocardial biopsy, the gold standard for the diagnosis of the disease. The objective of this study was to measure the MR properties of collagen using ultrashort TE (UTE), a technique that can detect short T2* species. Experiments were performed in collagen solutions. Via a model of bi-exponential T2* with oscillation, a linear relationship (slope = 0.40 ± 0.01, R(2) = 0.99696) was determined between the UTE collagen signal fraction associated with these properties and the measured collagen concentration in solution. The UTE signal of protons in the collagen molecule was characterized as having a mean T2* of 0.75 ± 0.05 ms and a mean chemical shift of -3.56 ± 0.01 ppm relative to water at 7 T. The results indicated that collagen can be detected and quantified using UTE. A knowledge of the collagen signal properties could potentially be beneficial for the endogenous detection of myocardial fibrosis.

Stage-two surgery using collagen soft tissue grafts: clinical cases and ultrastructural analysis.

OBJECTIVE: To present the application of two different soft tissue grafts around dental implants during stage-two surgery. Furthermore, the ultrastructure of these materials is shown and discussed using scanning electron microscopy (SEM). SUMMARY: Although soft tissue autografts may be currently regarded as the gold standard, harvesting of these grafts might lead to higher morbidity, longer chair time, and intra-/postoperative complications at the donor site. New developments in collagen scaff olds have provided an alternative to successfully replace autologous grafts in clinical practice. The SEM pictures clearly show the different composition of a bilayer scaff old (collagen matrix, CM) and a porcine acellular dermal matrix (ADM). These distinctive properties lead to different possible indications. Within the presented cases, ADM was used to augment the ridge contour and was placed into a buccal pouch to achieve complete coverage and an uneventful closed healing. On the other side, CM was left exposed to the oral cavity to successfully gain keratinized mucosa around and between two dental implants.

Vascular Ehlers-Danlos syndrome mutations in type III collagen differently stall the triple helical folding.

Vascular Ehlers-Danlos syndrome (EDS) type IV is the most severe form of EDS. In many cases the disease is caused by a point mutation of Gly in type III collagen. A slower folding of the collagen helix is a potential cause for over-modifications. However, little is known about the rate of folding of type III collagen in patients with EDS. To understand the molecular mechanism of the effect of mutations, a system was developed for bacterial production of homotrimeric model polypeptides. The C-terminal quarter, 252 residues, of the natural human type III collagen was attached to (GPP)7 with the type XIX collagen trimerization domain (NC2). The natural collagen domain forms a triple helical structure without 4-hydroxylation of proline at a low temperature. At 33 °C, the natural collagenous part is denatured, but the C-terminal (GPP)7-NC2 remains intact. Switching to a low temperature triggers the folding of the type III collagen domain in a zipper-like fashion that resembles the natural process. We used this system for the two known EDS mutations (Gly-to-Val) in the middle at Gly-910 and at the C terminus at Gly-1018. In addition, wild-type and Gly-to-Ala mutants were made. The mutations significantly slow down the overall rate of triple helix formation. The effect of the Gly-to-Val mutation is much more severe compared with Gly-to-Ala. This is the first report on the folding of collagen with EDS mutations, which demonstrates local delays in the triple helix propagation around the mutated residue.

Differences in collagen distribution of healthy and regenerated periodontium. Histomorphometric study in dogs.

Previous studies have shown that there is a relationship between periodontal disease and the distribution of collagen fibers. This study evaluated the distribution of collagen types I and III in regenerated bone and periodontal ligament, comparing them to the tissues near the regenerated area and to the healthy periodontium. In the third (P3) and fourth (P4) mandibular premolars of 5 healthy mongrel dogs, bilaterally, buccal class 2 furcation lesions were surgically created and chronified for 3 weeks. After that, full flaps were elevated and expanded polytetrafluoroethylene (e-PTFE) membranes were adapted, sutured and recovered by the flaps. Two weeks after surgery, two membranes on the same side were removed and the other membranes were removed four weeks after surgery. The dogs were euthanized at 12 weeks following placement of the e-PTFE membranes. P3 and P4 teeth as well as the second premolars (healthy control teeth) and their periodontal tissues were removed and histologically processed for Collagen Quantification (COLQ). The amount of type III collagen was higher in native bone compared to the regenerated area. For periodontal ligament, COLQ for type I collagen showed statistically significant differences (Tukeys's Multiple Comparison, p⟨0.05) between the regenerated groups and the control group. These differences were not found for type III COLQ. There are significant differences in collagen distribution among the regenerated, native and control tissues. Membrane removal 2 or 4 weeks postoperatively did not influence the collagen composition.

Effect of a collagen membrane combined with a porous titanium membrane on exophytic new bone formation in a rabbit calvarial model.

BACKGROUND: Previous studies showed that the use of a porous titanium membrane (TM) for exophytic bone regeneration does not effectively inhibit the infiltration of undesired tissue. Therefore, this study examines the effect of resorbable collagen membranes, such as cross-linked type I collagen membrane (BA) and double-layered porcine collagen membrane (BG), on the promotion of exophytic bone formation in guided bone regeneration when used in conjunction with a porous TM. METHODS: Thirty-six male New Zealand white rabbits were used in this study. Six rabbits were allotted to each test group. After decorticating the parietal bone, with or without filling the inner space with a freeze-dried cortical bone allograft (OG), the collagen membranes were fixed with metal pins. The experimental groups were divided into the following six groups: TM only, TM + OG, TM + BA, TM + BG, TM + OG + BA, and TM + OG + BG. The experimental animals were sacrificed at 8 and 16 weeks after surgery. Non-decalcified specimens were prepared and processed for histologic observations. The newly formed bone (percentage) was measured histomorphometrically. RESULTS: BG combined with TM promoted new bone formation and maturation by inhibiting the infiltration of connective tissue. However, BA had no significant effect on new bone formation. The amount of new bone formation was higher at 16 weeks than at 8 weeks, but the difference was not significant. At 16 weeks, the best result for newly formed bone was with TM + OG + BG, with a significant difference from TM alone and TM + BA. CONCLUSIONS: Regardless of the use of graft materials, BG combined with TM promoted more bone formation than BA combined with TM or TM alone. Thus, using a commercial collagen membrane to cover a TM can promote new exophytic bone formation.

Structural basis of fibrillar collagen trimerization and related genetic disorders.

The C propeptides of fibrillar procollagens have crucial roles in tissue growth and repair by controlling both the intracellular assembly of procollagen molecules and the extracellular assembly of collagen fibrils. Mutations in C propeptides are associated with several, often lethal, genetic disorders affecting bone, cartilage, blood vessels and skin. Here we report the crystal structure of a C-propeptide domain from human procollagen III. It reveals an exquisite structural mechanism of chain recognition during intracellular trimerization of the procollagen molecule. It also gives insights into why some types of collagen consist of three identical polypeptide chains, whereas others do not. Finally, the data show striking correlations between the sites of numerous disease-related mutations in different C-propeptide domains and the degree of phenotype severity. The results have broad implications for understanding genetic disorders of connective tissues and designing new therapeutic strategies.

Mapping of potent and specific binding motifs, GLOGEN and GVOGEA, for integrin α1ß1 using collagen toolkits II and III.

Integrins are well characterized cell surface receptors for extracellular matrix proteins. Mapping integrin-binding sites within the fibrillar collagens identified GFOGER as a high affinity site recognized by α2ß1, but with lower affinity for α1ß1. Here, to identify specific ligands for α1ß1, we examined binding of the recombinant human α1 I domain, the rat pheochromocytoma cell line (PC12), and the rat glioma Rugli cell line to our collagen Toolkit II and III peptides using solid-phase and real-time label-free adhesion assays. We observed Mg(2+)-dependent binding of the α1 I domain to the peptides in the following rank order: III-7 (GLOGEN), II-28 (GFOGER), II-7 and II-8 (GLOGER), II-18 (GAOGER), III-4 (GROGER). PC12 cells showed a similar profile. Using antibody blockade, we confirmed that binding of PC12 cells to peptide III-7 was mediated by integrin α1ß1. We also identified a new α1ß1-binding activity within peptide II-27. The sequence GVOGEA bound weakly to PC12 cells and strongly to activated Rugli cells or to an activated α1 I domain, but not to the α2 I domain or to C2C12 cells expressing α2ß1 or α11ß1. Thus, GVOGEA is specific for α1ß1. Although recognized by both α2ß1 and α11ß1, GLOGEN is a better ligand for α1ß1 compared with GFOGER. Finally, using biosensor assays, we show that although GLOGEN is able to compete for the α1 I domain from collagen IV (IC(50) ∼3 µm), GFOGER is much less potent (IC(50) ∼90 µm), as shown previously. These data confirm the selectivity of GFOGER for α2ß1 and establish GLOGEN as a high affinity site for α1ß1.

Disease-associated mutations prevent GPR56-collagen III interaction.

GPR56 is a member of the adhesion G protein-coupled receptor (GPCR) family. Mutations in GPR56 cause a devastating human brain malformation called bilateral frontoparietal polymicrogyria (BFPP). Using the N-terminal fragment of GPR56 (GPR56(N)) as a probe, we have recently demonstrated that collagen III is the ligand of GPR56 in the developing brain. In this report, we discover a new functional domain in GPR56(N), the ligand binding domain. This domain contains four disease-associated mutations and two N-glycosylation sites. Our study reveals that although glycosylation is not required for ligand binding, each of the four disease-associated mutations completely abolish the ligand binding ability of GPR56. Our data indicates that these four single missense mutations cause BFPP mostly by abolishing the ability of GPR56 to bind to its ligand, collagen III, in addition to affecting GPR56 protein surface expression as previously shown.

Proliferation assessment of primary human mesenchymal stem cells on collagen membranes for guided bone regeneration.

PURPOSE: Human mesenchymal stem cells (hMSCs) hold the potential for bone regeneration because of their self-renewing and multipotent character. The goal of this study was to evaluate the influence of collagen membranes on the proliferation of hMSCs derived from bone marrow. A special focus was set on short-term eluates derived from collagen membranes, as volatile toxic materials washed out from these membranes may influence cell behavior during the short time course of oral surgery. MATERIALS AND METHODS: The proliferation of hMSCs seeded directly on a collagen membrane (BioGide) was evaluated quantitatively using the cell proliferation reagent WST-1 (4-3-[4-iodophenyl]-2-[4-nitrophenyl]-2H-[5-tetrazolio]-1, 3--benzol-disulfonate) and qualitatively by scanning electron microscopy. Two standard biocompatibility tests, namely the lactate dehydrogenase and MTT (3-[4, 5-dimethyl-2-thiazolyl]-2, 5-diphenyl-2H-tetrazoliumbromide) tests, were performed using hMSCs cultivated in eluates from membranes incubated for 10 minutes, 1 hour, or 24 hours in serum-free cell culture medium. The data were analyzed statistically. RESULTS: Scanning electron microscopy showed large numbers of hMSCs with well-spread morphology on the collagen membranes after 7 days of culture. The WST test revealed significantly better proliferation of hMSCs on collagen membranes after 4 days of culture compared to cells cultured on a cover glass. Cytotoxicity levels were low, peaking in short-term eluates and decreasing with longer incubation times. CONCLUSION: Porcine collagen membranes showed good biocompatibility in vitro for hMSCs. If maximum cell proliferation rates are required, a prewash of membranes prior to application may be useful.

Autologous matrix-induced chondrogenesis aided reconstruction of a large focal osteochondral lesion of the talus.

The aim of this case report is to describe a novel technique for treatment of large osteochondral lesions of the talus using autologous matrix-induced chondrogenesis with a collagen I/III membrane.

Potential of skin fibroblasts for application to anterior cruciate ligament tissue engineering.

Fibroblasts isolated from skin and from anterior cruciate ligament (ACL) secrete type I and type III collagens in vivo and in vitro. However, it is much easier and practical to obtain a small skin biopsy than an ACL sample to isolate fibroblasts for tissue engineering applications. Various tissue engineering strategies have been proposed for torn ACL replacement. We report here the results of the implantation of bioengineered ACLs (bACLs), reconstructed in vitro using a type I collagen scaffold, anchored with two porous bone plugs to allow bone-ligament-bone surgical engraftment. The bACLs were seeded with autologous living dermal fibroblasts, and grafted for 6 months in goat knee joints. Histological and ultrastructural observations ex vivo demonstrated a highly organized ligamentous structure, rich in type I collagen fibers and cells. Grafts' vascularization and innervation were observed in all bACLs that were entirely reconstructed in vitro. Organized Sharpey's fibers and fibrocartilage, including chondrocytes, were present at the osseous insertion sites of the grafts. They showed remodeling and matrix synthesis postimplantation. Our tissue engineering approach may eventually provide a new solution to replace torn ACL in humans.