Abstract

Abstract

Osteoarthritis is a common articular cartilage disorder and causes a significant global disease burden. Articular cartilage has a limited capacity of repair and there is increasing interest in the use of cell-based therapies to facilitate repair including the use of Mesenchymal Stromal Cells (MSCs). There is some evidence in the literature that suggests that advancing age is associated with declining MSC function, including reduced proliferation and differentiation potential, and greater cellular apoptosis. In our study, we first performed a systematic review of the literature to determine the effects of chronological age on the in vitro properties of MSCs, and then performed a laboratory study to investigate these properties. We initially conducted a PRISMA systematic review of the literature to review the evidence base for the effects of chronological age on the in vitro properties of MSCs including cell numbers, expansion, cell surface characterization and differentiation potential. This was followed by laboratory based experiments to assess these properties. Tissue from patients undergoing total knee replacement surgery was used to isolate MSCs from the infrapatellar fat pad using a method developed in our laboratory. The growth kinetics was determined by calculating the population doublings per day. Following expansion in culture, MSCs at P2 were characterised for a panel of cell surface markers using flow cytometry. The cells were positive for CD73, CD90 and CD105, and negative for CD34 and CD45. The differentiation potential of the MSCs was assessed through tri-lineage differentiation assays. Chronological age-related changes in MSC function have important implications on the use of these cells in clinical applications for an ageing population. The results from this study will be used to plan further work looking at the effects of chronological age on cellular senescence and identify pathways that could be targeted to potentially reverse any age-related changes.

Abstract

Focal articular cartilage defects do not heal and, left untreated, progress to more widespread degenerative changes. A promising new approach for the repair of articular cartilage defects is the application of cell-based regenerative therapies using mesenchymal stromal cells (MSCs). MSCs are however present in a number of tissues and studies suggest that they vary in their proliferation, cell surface characterisation and differentiation. As the phenotypic properties of MSCs vary depending on tissue source, a systematic comparison of the transcriptomic signature would allow a better understanding of these differences between tissues, and allow the identification of markers specific to a MSC source that is best suited for clinical application. Tissue was used from patients undergoing total knee replacement surgery for osteoarthritis following ethical approval and informed consent. MSCs were isolated from bone, cartilage, synovium and infrapatellar fat pad. MSC number and expansion were quantified. Following expansion in culture, MSCs were characterised using flow cytometry with several cell surface markers; the cells from all sources were positive for CD44, CD90 and CD105. Their differentiation potential was assessed through tri-lineage differentiation assays. In addition, bulk mRNA-sequencing was used to determine the transcriptomic signatures. Differentially expressed (DE) genes were predicted. An enrichment analysis focused on the DE genes, against GO and pathway databases (KEGG and Reactome) was performed; protein-protein interaction networks were also inferred (Metascape, Reactome, Cytoscape). Optimal sourcing of MSCs will amplify their cartilage regeneration potential. This is imperative for assessing future therapeutic transplantation to maximise the chance of successful cartilage repair. A better understanding of differences in MSCs from various sources has implications beyond cartilage repair.

Abstract

Abstract

The aim of this study was to evaluate the role of peripheral blood derived mononucleated cells (PBMC) in osteochondral repair. We compared the healing of a critical size osteochondral defect in the medial femoral condyle and lateral trochlear sulcus in an ovine model.

Porous collagen-glycosaminoglycan (Col/GAG) scaffolds have previously been used clinically as regeneration templates for peripheral nerves and skin^[1]. For defects involving even minimal load-bearing applications however, these scaffolds do not possess the required stiffness. Calcium phosphates (CaPs) are often used as bone-graft substitutes due to their biocompatibility and direct bone-bonding ability. While CaPs have sufficient stiffness for bone-defect applications, unlike Col/GAG they lack elasticity and are very brittle. Combining these two materials produces a composite with enhanced material properties and chemical similarity to natural bone. The addition of CaP nanocrystallites into the Col/GAG matrix produces a 3-dimensional structure that maintains its structural integrity even when wet. In this study, the in vivo performance of mineralised Col/GAG composites was evaluated by implantation into a six-week ovine bone-defect model.

Four different materials were implanted; Col/GAG alone, Col/GAG with octacalcium phosphate, Col/GAG with hydroxyapatite and Col/GAG with brushite. Implants with a diameter of 9mm and length of 9mm, were placed bilaterally into the distal femoral condyle of the hind legs of thirteen sheep. This site was selected due to the large volume of load-bearing cancellous bone. Cancellous autograft was harvested from the tibial tuberosity and placed in the defect sites of two sheep as a positive control.

All animals were sacrificed after 6 weeks and tissue containing the implants was prepared for histological evaluation. Image analysis of Von Kossa stained sections showed that all mineralised Col/GAG implants had significantly more bone in the implant site than unmineralised Col/GAG but were not significantly different between CaPs. Interestingly, new bone formation often followed the structure of the porous material struts which acted as a template. The defect containing the autograft contained the greatest amount of new bone.

The aseptic loss of bone after hip replacement is a serious problem leading to implant instability. Hydroxyapatite coating of joint replacement components produces a bond with bone and helps to reduce loosening. However, over time bone remodeling at the implant interface leads to loss of hydroxyapatite. One possible solution would be to develop a coating that reduces hydroxyapatite and bone loss. Hydroxyapatite can be chemically modified through the substitution of ions to alter the biological response. Zinc is an essential trace element that has been found to inhibit osteoclast-like cell formation and decrease bone resorption. It was hoped that by substituting zinc into the hydroxyapatite lattice, the resultant zinc-substituted hydroxyapatite (ZnHA) would inhibit ceramic resorption and the resorption of bone. The aim of this work was to investigate the effect of ZnHA on the number and activity of osteoclasts.

Discs of phase pure hydroxyapatite (PPHA), 0.37wt% ZnHA and 0.58wt% ZnHA were produced, sintered at 1100 degrees Celsius and ground with 1200 grit silicon carbide paper. They were cultured in medium containing macrophage colony stimulating factor and receptor activator of nuclear factor kappa B ligand (RANKL) for 11 and 21 days. A control disc of PPHA cultured in medium containing no RANKL was also used. On the required dates the discs were removed and the cells stained for actin with phalloidin-TRITC and the cell nuclei with 4',6-Diamidino-2-phenylindole dihydrochloride. Cells with 3 or more nuclei were classed as osteoclasts and counted using ImageJ. On day 21 after the cells had been counted, the cells were removed and the discs coated in platinum before viewing with a scanning electron microscope. Resorption areas were then measured using ImageJ.

The addition of zinc was observed to significantly decrease the number of differentiated osteoclasts after 21 days (p<0.005 for 0.58wt% ZnHA compared to PPHA and p<0.01 for 0.37wt% ZnHA compared to PPHA). The area of resorption was also significantly decreased with the addition of zinc (p<0.005 for the comparison of 0.58wt% ZnHA with PPHA)

The work found that zinc substituted hydroxyapatite reduced the number and subsequent activity of osteoclasts.

Problems associated with allograft are well known. The addition of hydroxyapatite (HA) to allograft has various mechanical advantages, especially within revision arthroplasty. The mixing of bone and HA results in mechanical properties different from the individual parts. However, at present the changes in material properties the mix have not been fully investigated and the optimum mixing ratio not characterized. A compressive uniaxial chamber was used to investigate the change in mechanical properties occurring with the addition of HA in varying proportions to morcellised bone graft (MBG).

Carbonate-substituted hydroxyapatite (CHA) is more osteoconductive and more resorbable than hydroxyapatite (HA), but the underlying mode of its action is unclear. We hypothesised that increased resorption of the ceramic by osteoclasts might subsequently upregulate osteoblasts by a coupling mechanism, and sought to test this in a large animal model.

Defects were created in both the lateral femoral condyles of 12 adult sheep. Six were implanted with CHA granules bilaterally, and six with HA. Six of the animals in each group received the bisphosphonate zoledronate (0.05 mg/kg), which inhibits the function of osteoclasts, intra-operatively.

After six weeks bony ingrowth was greater in the CHA implants than in HA, but not in the animals given zoledronate. Functional osteoclasts are necessary for the enhanced osteoconduction seen in CHA compared with HA.