Collagen materials are extensively used in regenerative medicine. However, they still present limitations such as a mono-domain composition and poor mechanical properties. On the other hand, tissue grafts overcome most of these limitations. In addition, the potential of tissue grafts in musculoskeletal tissue engineering has not been fully investigated. Herein, we ventured to assess the potential of a decellularised porcine peritoneum for musculoskeletal applications by comparing its characteristics with a commercial collagen scaffold employed in tendon. Results indicated that the porcine peritoneum had higher mechanical properties and a lower crosslinking ratio (p < 0.01). Furthermore, it presented a lower resistance to collagenase digestion, which suggests a faster remodelling in vivo of the tissue graft. Immunohistochemistry analysis showed a preserved and multicomponent structure in the porcine peritoneum contrary to the collagen matrix, confirming the multifunctional nature of the tissue graft. Regarding the cell-response assessment, tenocytes and ADSCs were able to grow on both materials, however, proliferation was enhanced by the porcine peritoneum (p<0.01). Immune response by THP-1 showed an acute inflammatory response by macrophages to the collagen matrix, contrary to that observed in the porcine peritoneum which triggered a mild reaction. The in-progress in vivo study in a rabbit tendon model will elucidate the potential of porcine peritoneum for tendon repair applications. The present study shows how the multifunctionality of the porcine peritoneum provides higher cytocompatibility than a mono-domain collagen matrix with human tenocytes and ADSC. Besides, its lower immune response in vitro suggests better remodelling after implantation

Summary. Tissue grafts fail to recapitulate native tendon function, imposing the need for development of functional regeneration strategies. Herein, we describe advancements in tendon repair and regeneration using functionalised natural and synthetic devices and scaffold-free cell-based therapies. Introduction. Tendon and ligament injuries constitute an unmet clinical need with approximately 100,000 new cases annually in US alone. Tissue grafts are considered the gold standard in clinical practice. However, allografts and xenografts can lead to potential disease transmission, whilst the limited supply of autografts in severe injuries and degenerative conditions restricts their use. To this end, scaffold and scaffold-free therapies are under development to address the tissue grafts shortage. Herein, we describe biophysical, biochemical and biological methods to maintain tendon derived cell phenotype and/or differentiation of other cell types towards tenogenic lineage; development of tendon-equivalent facsimiles; and ultimately functional neotendon formation. Materials and Methods. Growth factor supplementation was assessed as means to either maintain tendon derived stem cell phenotype or differentiate them towards tenocytes. The influence of conditioning media was assessed as means to differentiate skin fibroblasts and stem cells towards tenogenic lineage. Biophysical and biochemical/biological features were assessed as means to maintain tendon derived cell phenotype and directional neotissue formation in rat patellar tendon model. Rich in tendon-specific extracellular matrix cell sheets were produced by appropriate modulation of the in vitro microenvironment. Structural, biophysical and biological analyses were subsequently carried out. Discussion & Future Studies. Treatment with 10 and 100 ng/mL of IGF-1 preserved tendon stem cell multipotency for up to 28 days in culture and minimised changes in marker expression and extracellular matrix molecules production enhancing that way the clinical potential of these cells. Hierarchically assembled collagen scaffolds and anisotropically ordered polymeric substrates of rigidity similar to native tendons facilitate tenocyte phenotype maintenance in vitro, whilst in vivo studies are under way to assess the extent of functional tendon regeneration. Appropriate modulation of the in vitro microenvironment of tenocytes with macromolecules enhances tendon specific extracellular matrix deposition within 6 days in culture, facilitating that way the wide acceptance of cell-sheet technology for tendon repair and regeneration

Abstract. Objectives. The patella tendon (PT) is commonly used as a graft material for anterior cruciate ligament reconstruction (ACLR). The function of the graft is to restore the mechanical behaviour of the knee joint. Therefore, it is essential that a robust methodology be developed for the mechanical testing of the PT, as well as for the tissue engineered grafts derived from this tissue. Our objectives were to (1) survey the literature, in order to define the state-of-the-art in mechanical testing of the PT, highlighting the most commonly used testing protocols, and (2) conduct validation studies using porcine PT to compare the mechanical measurements obtained using different methodological approaches. Methods. A PubMed search was performed using a boolean search term to identify publications consisting of PT tensile testing, and limited to records published in the past ten years (2010–2020). This returned a total of 143 publications. A meta-analysis was undertaken to quantify the frequency of commonly used protocol variations (pre-conditioning regime, strain rates, maximum strain, etc.). Validation studies were performed on porcine PT (n=4) using Instron tensile testing apparatus to examine the effect of preconditioning on low-strain (toe-region) mechanical properties. Results. Ramp-to-failure testing was found to be most commonly performed (included in over 90 % of publications), followed by stress relaxation and cyclic testing (∼25 %). Preconditioning was most commonly cyclic (27 %), involving 10–100 cycles. Validation studies show the number of cycles and duration of preconditioning, has no significant effect on toe region transition strain, transition stress, or sensitivity to increasing strain. Conclusions. There is a lack of standardisation in the mechanical testing of PT, which could have implications for the comparison of studies conducted using different protocols. However, variations in preconditioning regime have no effect on low-strain mechanical properties

Blood transfusion, organ and bone marrow transplantation and allogeneic tissue grafting create the potential for significant immunological challenges through the introduction of non-genetically identical major (HLA) and minor histocompatibility antigens (“allo-antigens”) into the body. Strategies to avoid the complications of immune responses against allo-antigens (transfusion reactions, rejection and graft versus host disease) include HLA matching, immunosuppressive therapies and immune tolerance promoting protocols. In the case of allogeneic mesenchymal stem/stromal cells (allo-MSC), it was initially believed that their combined properties of low HLA expression and inherent immune modulatory functions would render them invisible to the host immune system and, therefore, capable of being permanently accepted without further interventions. For clinical indications such as bone and tendon repair, in which permanent engraftment of allo-MSC or MSC-derived tissue constructs is particularly desirable, this model of “immune privilege” seemed almost too good to be true – and indeed, a decade of experimental research in this area has now convincingly demonstrated that allo-MSC typically elicit cellular (T-cell) and humoral (B-cell/antibody) immune responses in immunocompetent hosts – raising concern about their safety and long-term efficacy in human conditions. However, questions related to the immunogenicity of allo-MSC have evolved beyond a simple yes/no scenario to involve interesting observations and concepts about the potency, diversity, duration, functional characteristics and even potential clinical benfits of immunological responses to allo-MSC. In this presentation, I will summarise and critically evaluate current understanding of allo-MSC immunogenicity under experimental and clinical trial conditions with an emphasis on the implications for orthopaedic therapeutics

Summary Statement. A biomimetic tissue engineering strategy involving culture on bone scaffolds in perfusion bioreactors allows the construction of stable, viable, patient-specific bone-like substitutes from human induced pluripotent stem cells. Introduction. Tissue engineering of viable bone substitutes represents a promising therapeutic strategy to mitigate the burden of bone deficiencies. Human induced pluripotent stem cells (hiPSCs) have an excellent proliferation and differentiation capacity, and represent an unprecedented resource for engineering of autologous tissue grafts, as well as advanced tissue models for biological studies and drug discovery. A major challenge is to reproducibly expand, differentiate and organize hiPSCs into mature, stable tissue structures. Based on previous studies (1,2,3), we hypothesised that the culture conditions supporting bone tissue formation from adult human mesenchymal stem cells (hMSCs) and human embryonic stem cell (hESC)-derived mesenchymal progenitors could be translated to hiPSC-derived mesenchymal progenitors. Our objectives were to: 1. Derive and characterise mesenchymal progenitors from hiPSC lines. 2. Engineer bone substitutes from progenitor lines exhibiting osteogenic potential in an osteoconductive scaffold – perfusion bioreactor culture model. 3. Assess the molecular changes associated with the culture of hiPSC-progenitors in perfusion bioreactors, and evaluate the stability of engineered bone tissue substitutes in vivo. Methods. hESC and hiPSC lines (derived using retroviral vectors, Sendai virus and episomal vectors) were karyotyped, characterised for pluripotency and induced into the mesenchymal lineage. Mesenchymal progenitors were evaluated for growth potential, expression of surface markers and differentiation potential. Progenitors exhibiting osteogenic potential were cultured on decellularised bovine bone scaffolds in perfusion bioreactors for 5 weeks as previously (3). Global gene expression profiles were evaluated prior and after bioreactor culture. Bone development was investigated using biochemical and histological methods, and by micro-computed tomography (μCT) imaging over the duration of bioreactor culture and after 12-week subcutaneous implantation in immunodeficient mice. Results. Progenitors with high proliferation potential, expressing typical mesenchymal surface antigens were successfully derived from three hiPSC lines. Differences in mesenchymal surface antigens expression and global gene expression profiles of progenitors from different lines corresponded to their differentiation abilities toward the osteogenic, chondrogenic and adipogenic lineages. Bioreactor culture yielded constructs with significantly higher cellularity, AP activity and osteopontin release into the culture medium as compared to static culture. Dense bone matrix formation was evidenced by the positive staining of collagen, osteopontin, bone sialoprotein and osteocalcin. In comparison, static culture yielded constructs with uniformly distributed cells, however tissue formation was scarce. μCT revealed a significant increase in bone structural parameters, evidencing mineralization of the deposited bone tissue during the 5-week culture in bioreactors. Osteogenesis and bone tissue formation were comparable between hESCs, hiPSCs and hMSCs (3). Bioreactor cultivation resulted in repression of genes involved in proliferation and tumorigenesis, and upregulation of genes associated with osteogenesis and bone development. Engineered bone tissue displayed stable phenotype after 12-week implantation in vivo, with cells of human origin, ingrowing vasculature and osteoclasts, suggesting an initiation of tissue remodeling. Discussion/Conclusion. Our biomimetic strategy opens the possibility to construct an unlimited quantity of patient-specific bone grafts for personalised applications, and to generate qualified experimental models to study bone biology under normal and pathological conditions, as well as test new drugs using selected pools of hiPSC lines

We hypothesised that meniscal tears treated with mesenchymal stem cells (MSCs) together with a conventional suturing technique would show improved healing compared with those treated by a conventional suturing technique alone. In a controlled laboratory study 28 adult pigs (56 knees) underwent meniscal procedures after the creation of a radial incision to represent a tear. Group 1 (n = 9) had a radial meniscal tear which was left untreated. In group 2 (n = 19) the incision was repaired with sutures and fibrin glue and in group 3, the experimental group (n = 28), treatment was by MSCs, suturing and fibrin glue.

At eight weeks, macroscopic examination of group 1 showed no healing in any specimens. In group 2 no healing was found in 12 specimens and incomplete healing in seven. The experimental group 3 had 21 specimens with complete healing, five with incomplete healing and two with no healing. Between the experimental group and each of the control groups this difference was significant (p < 0.001).

The histological and macroscopic findings showed that the repair of meniscal tears in the avascular zone was significantly improved with MSCs, but that the mechanical properties of the healed menisci remained reduced.

We attempted to repair full-thickness defects in the articular cartilage of the trochlear groove of the femur in 30 rabbit knee joints using allogenic cultured chondrocytes embedded in a collagen gel. The repaired tissues were examined at 2, 4, 8, 12 and 24 weeks after operation using histological and histochemical methods. The articular defect filling index measurement was derived from safranin-O stained sections. Apoptotic cellular fractions were derived from analysis of apoptosis in situ using TUNEL staining, and was confirmed using caspase-3 staining along with quantification of the total cellularity. The mean articular defect filling index decreased with time. After 24 weeks it was 0.7 (sd 0.10), which was significantly lower than the measurements obtained earlier (p < 0.01). The highest mean percentage of apoptotic cells were observed at 12 weeks, although the total cellularity decreased with time. Because apoptotic cell death may play a role in delamination after chondrocyte transplantation, anti-apoptotic gene therapy may protect transplanted chondrocytes from apoptosis.

Impacted bone allograft is often used in revision joint replacement. Hydroxyapatite granules have been suggested as a substitute or to enhance morcellised bone allograft. We hypothesised that adding osteogenic protein-1 to a composite of bone allograft and non-resorbable hydroxyapatite granules (ProOsteon) would improve the incorporation of bone and implant fixation. We also compared the response to using ProOsteon alone against bone allograft used in isolation. We implanted two non-weight-bearing hydroxyapatite-coated implants into each proximal humerus of six dogs, with each implant surrounded by a concentric 3 mm gap. These gaps were randomly allocated to four different procedures in each dog: 1) bone allograft used on its own; 2) ProOsteon used on its own; 3) allograft and ProOsteon used together; or 4) allograft and ProOsteon with the addition of osteogenic protein-1.

After three weeks osteogenic protein-1 increased bone formation and the energy absorption of implants grafted with allograft and ProOsteon. A composite of allograft, ProOsteon and osteogenic protein-1 was comparable, but not superior to, allograft used on its own.

ProOsteon alone cannot be recommended as a substitute for allograft around non-cemented implants, but should be used to extend the volume of the graft, preferably with the addition of a growth factor.