Benjamin B. Rothrauff

Tendon and ligament regeneration and repair: clinical relevance and developmental paradigm.

As dense connective tissues connecting bone to muscle and bone to bone, respectively, tendon and ligament (T/L) arise from the somitic mesoderm, originating in a recently discovered somitic compartment, the syndetome. Inductive signals from the adjacent sclerotome and myotome upregulate expression of Scleraxis, a key transcription factor for tenogenic and ligamentogenic differentiation. Understanding T/L development is critical to establishing a knowledge base for improving the healing and repair of T/L injuries, a high-burden disease due to the intrinsically poor natural healing response. Current treatment of the three most common tendon injuries-tearing of the rotator cuff of the shoulder, flexor tendon of the hand, and Achilles tendon-include mostly surgical repair and/or conservative approaches, including biophysical modalities such as rehabilitation and cryotherapy. Unfortunately, the fibrovascular scar formed during healing possesses inferior mechanical and biochemical properties, resulting in compromised tissue functionality. Regenerative approaches have sought to augment the injured tissue with cells, scaffolds, bioactive agents, and mechanical stimulation to improve the natural healing response. The key challenges in restoring full T/L function following injury include optimal combination of these biological agents as well as their delivery to the injury site. A greater understanding of the molecular mechanisms involved in T/L development and natural healing, coupled with the capability of producing complex biomaterials to deliver multiple biofactors with high spatiotemporal resolution and specificity, should lead to regenerative procedures that more closely recapitulate T/L morphogenesis, thereby offering future patients the prospect of T/L regeneration, as opposed to simple tissue repair.

Enhancement of Tenogenic Differentiation of Human Adipose Stem Cells by Tendon-Derived Extracellular Matrix

Mesenchymal stem cells (MSCs) have gained increasing research interest for their potential in improving healing and regeneration of injured tendon tissues. Developing functional three-dimensional (3D) scaffolds to promote MSC proliferation and differentiation is a critical requirement in tendon tissue engineering. Tendon extracellular matrix has been shown to maintain the tenogenic potential of tendon stem cells and stimulate tenogenesis of human adipose stem cells (hASCs) in 2D culture. This study aims at characterizing the biological composition of urea-extracted fraction of tendon ECM (tECM) and its tenogenic effect on hASCs cultured in a 3D collagen scaffold under uniaxial tension. The tECM obtained was cell-free and rich in ECM proteins. hASCs seeded in tECM-supplemented scaffold exhibited significantly increased proliferation and tenogenic differentiation. The presence of tECM also greatly suppressed the osteogenic differentiation of hASCs triggered by uniaxial tension. In addition, tECM-supplemented constructs displayed enhanced mechanical strength, accompanied by reduced expression and activity of MMPs in the seeded hASCs, indicating a regulatory activity of tECM in cell-mediated scaffold remodeling. These findings support the utility of tECM in creating bio-functional scaffolds for tendon tissue engineering.

The Structure and Function of the Anterolateral Ligament of the Knee: A Systematic Review

PURPOSE The purpose of this systematic review was to evaluate the anatomic structure and function of the anterolateral ligament (ALL) of the knee. METHODS The Medline, Embase, and Cochrane databases were screened for all studies related to the ALL of the knee. Two reviewers independently reviewed all eligible articles and the references of these articles. Inclusion and exclusion criteria were applied to all searched studies. Quality assessment was completed for the included studies. RESULTS Nineteen studies were identified for final analysis. Pooled analysis identified the ALL in 430 of 449 knees (96%) examined. The ligament was found to originate from the region of the lateral femoral epicondyle and insert on the proximal tibia midway between the Gerdy tubercle and the fibular head. The ALL was found to be 34.1 to 41.5 mm in length, 5.1 to 8.3 mm in width above the lateral meniscus, and 8.9 to 11.2 mm in width below the lateral meniscus. By use of magnetic resonance imaging, the ALL was identified in 93% of knees examined (clinical, 64 of 70; cadaveric, 16 of 16). In one case study the ligament was clearly visualized by ultrasound examination. Histologic analysis across 3 studies showed characteristics consistent with ligamentous tissue. Though not shown in biomechanical studies, it is hypothesized that the ALL provides anterolateral stability to the knee, preventing anterolateral subluxation of the proximal tibia on the femur. One study identified a network of peripheral nerves, suggesting a proprioceptive function of the ALL. CONCLUSIONS This systematic review shows the ALL to be a distinct structure with a consistent origin and insertion sites. The ALL is an extra-articular structure with a clear course from the lateral femoral epicondyle region, running anteroinferiorly, to the proximal tibia at a site midway between the Gerdy tubercle and the head of the fibula. The function of this ligament is theorized to provide anterolateral knee stability. LEVEL OF EVIDENCE Level IV, systematic review of cadaveric and imaging studies.

Multilayered polycaprolactone/gelatin fiber-hydrogel composite for tendon tissue engineering.

UNLABELLED Regeneration of injured tendon and ligament (T&L) remains a clinical challenge due to their poor intrinsic healing capacity. Tissue engineering provides a promising alternative treatment approach to facilitate T&L healing and regeneration. Successful tendon tissue engineering requires the use of three-dimensional (3D) biomimetic scaffolds that possess the physical and biochemical features of native tendon tissue. We report here the development and characterization of a novel composite scaffold fabricated by co-electrospinning of poly-ε-caprolactone (PCL) and methacrylated gelatin (mGLT). We found that photocrosslinking retained mGLT, resulted in a uniform distribution of mGLT throughout the depth of scaffold and also preserved scaffold mechanical strength. Moreover, photocrosslinking was able to integrate stacked scaffold sheets to form multilayered constructs that mimic the structure of native tendon tissues. Importantly, cells impregnated into the constructs remained responsive to topographical cues and exogenous tenogenic factors, such as TGF-β3. The excellent biocompatibility and highly integrated structure of the scaffold developed in this study will allow the creation of a more advanced tendon graft that possesses the architecture and cell phenotype of native tendon tissues. STATEMENT OF SIGNIFICANCE The clinical challenges in tendon repair have spurred the development of tendon tissue engineering approaches to create functional tissue replacements. In this study, we have developed a novel composite scaffold as a tendon graft consisting of aligned poly-ε-caprolactone (PCL) microfibers and methacrylated gelatin (mGLT). Cell seeding and photocrosslinking between scaffold layers can be performed simultaneously to create cell impregnated multilayered constructs. This cell-scaffold construct combines the advantages of PCL nanofibrous scaffolds and photocrosslinked gelatin hydrogels to mimic the structure, mechanical anisotropy, and cell phenotype of native tendon tissue. The scaffold engineered here as a building block for multilayer constructs should have applications beyond tendon tissue engineering in the fabrication of tissue grafts that consist of both fibrous and hydrogel components.

Aging of the skeletal muscle extracellular matrix drives a stem cell fibrogenic conversion

Age‐related declines in skeletal muscle regeneration have been attributed to muscle stem cell (MuSC) dysfunction. Aged MuSCs display a fibrogenic conversion, leading to fibrosis and impaired recovery after injury. Although studies have demonstrated the influence of in vitro substrate characteristics on stem cell fate, whether and how aging of the extracellular matrix (ECM) affects stem cell behavior has not been investigated. Here, we investigated the direct effect of the aged muscle ECM on MuSC lineage specification. Quantification of ECM topology and muscle mechanical properties reveals decreased collagen tortuosity and muscle stiffening with increasing age. Age‐related ECM alterations directly disrupt MuSC responses, and MuSCs seeded ex vivo onto decellularized ECM constructs derived from aged muscle display increased expression of fibrogenic markers and decreased myogenicity, compared to MuSCs seeded onto young ECM. This fibrogenic conversion is recapitulated in vitro when MuSCs are seeded directly onto matrices elaborated by aged fibroblasts. When compared to young fibroblasts, fibroblasts isolated from aged muscle display increased nuclear levels of the mechanosensors, Yes‐associated protein (YAP)/transcriptional coactivator with PDZ‐binding motif (TAZ), consistent with exposure to a stiff microenvironment in vivo. Accordingly, preconditioning of young fibroblasts by seeding them onto a substrate engineered to mimic the stiffness of aged muscle increases YAP/TAZ nuclear translocation and promotes secretion of a matrix that favors MuSC fibrogenesis. The findings here suggest that an age‐related increase in muscle stiffness drives YAP/TAZ‐mediated pathogenic expression of matricellular proteins by fibroblasts, ultimately disrupting MuSC fate.

Anatomical region-dependent enhancement of 3-dimensional chondrogenic differentiation of human mesenchymal stem cells by soluble meniscus extracellular matrix

Extracellular matrix (ECM) derived from decellularized tissues has been found to promote tissue neogenesis, most likely mediated by specific biochemical and physical signaling motifs that promote tissue-specific differentiation of progenitor cells. Decellularized ECM has been suggested to be efficacious for the repair of tissue injuries. However, decellularized meniscus contains a dense collagenous structure, which impedes cell seeding and infiltration and is not readily applicable for meniscus repair. In addition, the meniscus consists of two distinct anatomical regions that differ in vascularity and cellular phenotype. The purpose of this study was to explore the region-specific bioactivity of solubilized ECM derived from the inner and outer meniscal regions as determined in 2D and 3D cultures of adult mesenchymal stem cells (MSCs). When added as a medium supplement to 2D cultures of MSCs, urea-extracted fractions of the inner (imECM) and outer meniscal ECM (omECM) enhanced cell proliferation while imECM most strongly upregulated fibrochondrogenic differentiation on the basis of gene expression profiles. When added to 3D cultures of MSCs seeded in photocrosslinked methacrylated gelatin (GelMA) hydrogels, both ECM fractions upregulated chondrogenic differentiation as determined by gene expression and protein analyses, as well as elevated sulfated glycosaminoglycan sGAG content, compared to ECM-free controls. The chondrogenic effect at day 21 was most pronounced with imECM supplementation, but equivalent between ECM groups by day 42. Despite increased cartilage matrix, imECM and omECM constructs possessed compressive moduli similar to controls. In conclusion, soluble meniscal ECM may be considered for use as a tissue-specific reagent to enhance chondrogenesis for MSC-based 3D cartilage tissue engineering. STATEMENT OF SIGNIFICANCE The inner region of the knee meniscus is frequently injured and possesses a poor intrinsic healing capacity. Solubilized extracellular matrix (ECM) derived from decellularized meniscus tissue may promote homologous differentiation of progenitor cells, thereby enhancing fibrocartilage formation within a meniscal lesion. However, the meniscus possesses regional variation in ultrastructure, biochemical composition, and cell phenotype, which may affect the bioactivity of soluble ECM derived from different regions of decellularized menisci. In this study, we demonstrate that urea-extracted fractions of ECM derived from the inner and outer regions of menisci enhance chondrogenesis in mesenchymal stem cells seeded in 3-dimensional photocrosslinkable hydrogels and that this effect is more strongly mediated by inner meniscal ECM. These findings suggest region-specific bioactivity of decellularized meniscal ECM.

Region-Specific Effect of the Decellularized Meniscus Extracellular Matrix on Mesenchymal Stem Cell-Based Meniscus Tissue Engineering.

Background: The meniscus is the most commonly injured knee structure, and surgical repair is often ineffective. Tissue engineering–based repair or regeneration may provide a needed solution. Decellularized, tissue-derived extracellular matrices (ECMs) have received attention for their potential use as tissue-engineered scaffolds. In considering meniscus-derived ECMs (mECMs) for meniscus tissue engineering, it is noteworthy that the inner and outer regions of the meniscus have different structural and biochemical features, potentially directing the differentiation of cells toward region-specific phenotypes. Purpose: To investigate the applicability of mECMs for meniscus tissue engineering by specifically comparing region-dependent effects of mECMs on 3-dimensional constructs seeded with human bone marrow mesenchymal stem cells (hBMSCs). Study Design: Controlled laboratory study. Methods: Bovine menisci were divided into inner and outer halves and were minced, treated with Triton X-100 and DNase, and extracted with urea. Then, hBMSCs (1 × 106 cells/mL) were encapsulated in a photo–cross-linked 10% polyethylene glycol diacrylate scaffold containing mECMs (60 μg/mL) derived from either the inner or outer meniscus, with an ECM-free scaffold as a control. The cell-seeded constructs were cultured with chondrogenic medium containing recombinant human transforming growth factor β3 (TGF-β3) and were analyzed for expression of meniscus-associated genes as well as for the collagen (hydroxyproline) and glycosaminoglycan content as a function of time. Results: Decellularization was verified by the absence of 4′,6-diamidino-2-phenylindole (DAPI)–stained cell nuclei and a reduction in the DNA content. Quantitative real-time polymerase chain reaction showed that collagen type I expression was significantly higher in the outer mECM group than in the other groups, while collagen type II and aggrecan expression was highest in the inner mECM group. The collagen (hydroxyproline) content was highest in the outer mECM group, while the glycosaminoglycan content was higher in both the inner and outer mECM groups compared with the control group. Conclusion: These results showed that the inner mECM enhances the fibrocartilaginous differentiation of hBMSCs, while the outer mECM promotes a more fibroblastic phenotype. Our findings support the feasibility of fabricating bioactive scaffolds using region-specific mECM preparations for meniscus tissue engineering. Clinical Relevance: This is the first report to demonstrate the feasibility of applying region-specific mECMs for the engineering of meniscus implants capable of reproducing the biphasic, anatomic, and biochemical characteristics of the meniscus, features that should contribute to the feasibility of their clinical application.

Effect of adipose-derived stromal cells and BMP12 on intrasynovial tendon repair: A biomechanical, biochemical, and proteomics study

The outcomes of flexor tendon repair are highly variable. As recent efforts to improve healing have demonstrated promise for growth factor‐ and cell‐based therapies, the objective of the current study was to enhance repair via application of autologous adipose derived stromal cells (ASCs) and the tenogenic growth factor bone morphogenetic protein (BMP) 12. Controlled delivery of cells and growth factor was achieved in a clinically relevant canine model using a nanofiber/fibrin‐based scaffold. Control groups consisted of repair‐only (no scaffold) and acellular scaffold. Repairs were evaluated after 28 days of healing using biomechanical, biochemical, and proteomics analyses. Range of motion was reduced in the groups that received scaffolds compared to normal. There was no effect of ASC + BMP12 treatment for range of motion or tensile properties outcomes versus repair‐only. Biochemical assays demonstrated increased DNA, glycosaminoglycans, and crosslink concentration in all repair groups compared to normal, but no effect of ASC + BMP12. Total collagen was significantly decreased in the acellular scaffold group compared to normal and significantly increased in the ASC + BMP12 group compared to the acellular scaffold group. Proteomics analysis comparing healing tendons to uninjured tendons revealed significant increases in proteins associated with inflammation, stress response, and matrix degradation. Treatment with ASC + BMP12 amplified these unfavorable changes. In summary, the treatment approach used in this study induced a negative inflammatory reaction at the repair site leading to poor healing. Future approaches should consider cell and growth factor delivery methods that do not incite negative local reactions.

Tissue-specific bioactivity of soluble tendon-derived and cartilage-derived extracellular matrices on adult mesenchymal stem cells

BackgroundBiological scaffolds composed of tissue-derived extracellular matrix (ECM) can promote homologous (i.e., tissue-specific) cell differentiation through preservation of biophysical and biochemical motifs found in native tissues. Solubilized ECMs derived from decellularized tendon and cartilage have recently been promoted as tissue-specific biomaterials, but whether tissue-specific bioactivity is preserved following solubilization is unknown. This study explored the tissue-specific bioactivity of soluble decellularized tendon and cartilage ECMs on human bone marrow-derived mesenchymal stem cells (MSCs) presented across different culture microenvironments, including two-dimensional (2D) tissue culture plastic, aligned electrospun nanofibers, cell pellets, and cell-seeded photocrosslinkable hydrogels.MethodsTendon and cartilage ECMs were decellularized using established methods and solubilized either via pepsin digestion or urea extraction. The effect of soluble ECMs on cell proliferation and differentiation was initially explored by supplementing basal medium of human MSCs cultured on 2D tissue culture plastic. In subsequent experiments, MSCs were cultured on aligned electrospun nanofibers, ascell pellets, or encapsulated within photocrosslinkable methacrylated gelatin (GelMA) hydrogels. Urea-extracted tendon and cartilage ECMs were added as supplements.ResultsPepsin-digested ECMs did not promote homologous differentiation in human MSCs, whether provided as a medium supplement or three-dimensional (3D) hydrogels. In contrast, urea-extracted ECMs tended to promote tissue-specific differentiation of MSCs cultured in 2D and 3D microenvironments. The application of the small molecule TGF-β signaling inhibitor SB-431542 largely negated the tissue-specific gene expression patterns mediated by tendon and cartilage ECMs. This suggests that the action of endogenous TGF-β was required, but was not sufficient, to impart tissue-specific bioactivity of urea-extracted ECMs. When urea-extracted cartilage ECM was incorporated within a photocurable GelMA hydrogel it independently enhanced chondrogenesis in encapsulated MSCs, and showed additive prochondrogenesis upon TGF-β supplementation in the medium.ConclusionsUrea-extracted ECM fractions of decellularized tendon and cartilage are soluble supplements capable of enhancing tissue-specific differentiation of adult stem cells.

Management of the Contaminated Anterior Cruciate Ligament Graft

PURPOSE This systematic review explores management strategies for intraoperative anterior cruciate ligament (ACL) graft contamination. METHODS Two databases (Medline and EMBASE) were screened for studies involving ACL graft contamination published between 1946 and April 2013. We included studies evaluating the management of a contaminated graft and excluded small case-series studies. We conducted a full-text review of eligible studies, and the references were searched for additional eligible studies. Inclusion and exclusion criteria were applied to the searched studies. RESULTS Our search yielded 6 laboratory investigations with a total of 495 graft samples used. These samples were contaminated and cleansed by various methods. The most successful sterilization protocols used chlorhexidine or mechanical agitation with a polymyxin B-bacitracin solution to achieve sterility in 100% of their respective experimental graft tissues. A chlorhexidine soak and plain bacitracin soak were also effective, at 97.5% and 97%, respectively. Povidone-iodine and an antibiotic soak of polymyxin-bacitracin were the least effective, with sterility rates of 48% and 57%, respectively. CONCLUSIONS The results of this review suggest that the optimal agent for sterilizing a dropped graft is chlorhexidine. A protocol of mechanical agitation and serial dilution with a polymyxin B-bacitracin solution was also highly effective; however, the sample size was too small to realistically recommend its use. Bacitracin alone was also found to be an effective sterilization agent, as was a combined solution of neomycin and polymyxin B. Pooled results showed that normal saline solution, povidone-iodine, and a polymyxin B-bacitracin solution all yielded suboptimal sterilization. The available evidence, however, is laboratory based and may not accurately reflect clinical conditions; moreover, there is a lack of biomechanical studies evaluating sterilized grafts. As a result, the findings should be interpreted with caution. LEVEL OF EVIDENCE Level IV, systematic review of basic science studies.