Adult articular cartilage mechanical functionality is dependent on the unique zonal organization of its tissue. Current mesenchymal stem cell (MSC)-based treatment has resulted in sub-optimal cartilage repair, with inferior quality of cartilage generated from MSCs in terms of the biochemical content, zonal architecture and mechanical strength when compared to normal cartilage. The phenotype of cartilage derived from MSCs has been reported to be influenced by the microenvironmental biophysical cues, such as the surface topography and substrate stiffness. In this study, the effect of nano-topographic surfaces to direct MSC chondrogenic differentiation to chondrocytes of different phenotypes was investigated, and the application of these pre-differentiated cells for cartilage repair was explored. Specific nano-topographic patterns on the polymeric substrate were generated by nano-thermal imprinting on the PCL, PGA and PLA surfaces respectively. Human bone marrow MSCs seeded on these surfaces were subjected to chondrogenic differentiation and the phenotypic outcome of the differentiated cells was analyzed by real time PCR, matrix quantification and immunohistological staining. The influence of substrate stiffness of the nano-topographic patterns on MSC chondrogenesis was further evaluated. The ability of these pre-differentiated MSCs on different nano-topographic surfaces to form zonal cartilage was verified in in vitro 3D hydrogel culture. These pre-differentiated cells were then implanted as bilayered hydrogel constructs composed of superficial zone-like chondro-progenitors overlaying the middle/deep zone-like chondro-progenitors, was compared to undifferentiated MSCs and non-specifically pre-differentiated MSCs in a osteochondral defect rabbit model. Nano-topographical patterns triggered MSC morphology and cytoskeletal structure changes, and cellular aggregation resulting in specific chondrogenic differentiation outcomes. MSC chondrogenesis on nano-pillar topography facilitated robust hyaline-like cartilage formation, while MSCs on nano-grill topography were induced to form fibro/superficial zone cartilage-like tissue. These phenotypic outcomes were further diversified and controlled by manipulation of the material stiffness. Hyaline cartilage with middle/deep zone cartilage characteristics was derived on softer nano-pillar surfaces, and superficial zone-like cartilage resulted on softer nano-grill surfaces. MSCs on stiffer nano-pillar and stiffer nano-grill resulted in mixed fibro/hyaline/hypertrophic cartilage and non-cartilage tissue, respectively. Further, the nano-topography pre-differentiated cells possessed phenotypic memory, forming phenotypically distinct cartilage in subsequent 3D hydrogel culture. Lastly, implantation of the bilayered hydrogel construct of superficial zone-like chondro-progenitors and middle/deep zone-like chondro-progenitors resulted in regeneration of phenotypically better cartilage tissue with higher mechanical function. Our results demonstrate the potential of nano-topographic cues, coupled with substrate stiffness, in guiding the differentiation of MSCs to chondrocytes of a specific phenotype. Implantation of these chondrocytes in a bilayered hydrogel construct yielded cartilage with more normal architecture and mechanical function. Our approach provides a potential translatable strategy for improved articular cartilage regeneration using MSCs

Osteoarthritis (OA) is a debilitating disease characterised by degradation of articular cartilage and subchondral bone remodeling. Current therapies for early or midstage disease do not regenerate articular cartilage, or fail to integrate the repair tissue with host tissue, and therefore there is great interest in developing biological approaches to cartilage repair. We have shown previously that platelet-rich plasma (PRP) can enhance cartilage tissue formation. PRP is obtained from a patient's own blood, and is an autologous source of many growth factors and other molecules which may aid in healing. This raised the question as to whether PRP could enhance cartilage integration. We hypothesise that PRP will enhance integration of bioengineered cartilage with native cartilage. Chondrocytes were isolated from bovine metacarpal-phalangeal joints, seeded on a porous bone substitute (calcium polyphosphate) and grown in the presence of FBS to form an in vitro model of osteochondral-like tissue. After 7 days, the biphasic constructs were soaked in PRP for 30 minutes prior to implantation into the core of a ring-shaped biphasic explant of native bovine cartilage and bone. Controls were not soaked in PRP. The resulting implant-explant construct was cultured in a stirring bioreactor in serum free conditions for 2 weeks. The integration zone was visualised histologically. A push-out test was performed to assess the strength of integration. Matrix accumulation at the zone of integration was assessed biochemically and the gene expression of the cells in this region was assessed by RT-PCR. Significance (p<0.05) was assessed by a student's t-test or one-way ANOVA with tukey's post hoc. PRP soaked bioengineered implants, integrated with the host tissue in 73% of samples, whereas control bioengineered implants only integrated in 19% of samples based on macroscopic evaluation (p<0.05). The integration strength, as determined by the normalised maximum force to failure, was significantly increased in the PRP soaked implant group compared to controls (219 +/− 35.4 kPa and 72.0 +/− 28.5 kPa, respectively, p<0.05). This correlated with an increase in glycosaminoglycan and collagen accumulation in the region of integration in the PRP treated implant group, compared to untreated controls after 2 weeks (p<0.05). Immunohistochemical studies revealed that the integration zone was rich in collagen type II and aggrecan. The cells at the zone of integration in the PRP soaked group had a 2.5 fold increase in aggrecan gene expression (p=0.05) and a 3.5 fold increase in matrix metalloproteinase 13 expression (p<0.05) compared to controls. PRP soaked bio-engineered cartilage implants showed improved integration with native cartilage compared to non-treated implants, perhaps due to the increased matrix accumulation and remodeling at the interface. Further evaluation is required to determine if PRP improves integration in vivo

Construction of a functional skeleton is accomplished through co-ordination of the developmental processes of chondrogenesis, osteogenesis, and synovial joint formation. Infants whose movement in utero is reduced or restricted and who subsequently suffer from joint dysplasia (including joint contractures) and thin hypo-mineralised bones, demonstrate that embryonic movement is crucial for appropriate skeletogenesis. This has been confirmed in mouse, chick, and zebrafish animal models, where reduced or eliminated movement consistently yields similar malformations and which provide the possibility of experimentation to uncover the precise disturbances and the mechanisms by which movement impacts molecular regulation. Molecular genetic studies have shown the important roles played by cell communication signalling pathways, namely Wnt, Hedgehog, and transforming growth factor-beta/bone morphogenetic protein. These pathways regulate cell behaviours such as proliferation and differentiation to control maturation of the skeletal elements, and are affected when movement is altered. Cell contacts to the extra-cellular matrix as well as the cytoskeleton offer a means of mechanotransduction which could integrate mechanical cues with genetic regulation. Indeed, expression of cytoskeletal genes has been shown to be affected by immobilisation. In addition to furthering our understanding of a fundamental aspect of cell control and differentiation during development, research in this area is applicable to the engineering of stable skeletal tissues from stem cells, which relies on an understanding of developmental mechanisms including genetic and physical criteria. A deeper understanding of how movement affects skeletogenesis therefore has broader implications for regenerative therapeutics for injury or disease, as well as for optimisation of physical therapy regimes for individuals affected by skeletal abnormalities.

Cite this article: Bone Joint Res 2015;4:105–116