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 and gender 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 and gender 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 and gender 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. Compare the extent of the effect of age on MSC cell marker expression, proliferation and pathways. Tissue from patients undergoing total knee replacement surgery was used to isolate MSCs from the synovium, fat pad and bone fragments 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 antibody cocktail (eg included CD34, CD45). The differentiation potential of the MSCs was assessed through tri-lineage differentiation assays. At P2 after extracting RNA, we investigate the gene analysis using Bulk seq. Clear differences between the younger and older patients and gender were indicated.Abstract
Objectives
Methods and Results
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
SOX genes comprise a family of transcription factors characterised by a conserved HMG-box domain that confer pleiotropic effects on cell fate and differentiation through binding to the minor groove of DNA. Paracrine regulation and contact-dependant Notch signalling has been suggested to modulate the induction of SOX gene expression. The objective of this study is to investigate the crosstalk between mesenchymal stromal cells (MSCs) and chondrocytes by comparing SOX gene expression in their co-culture and respective monocultures. Our study adopted an Abstract
Objective
Methods
Mesenchymal stem cells (MSCs) and chondrocytes have both been crucial in trials for cartilage repair, and there has been growing interest into their respective secretomes owing to their role in chondrogenic crosstalk. This has been studied by Our study utilised an Abstract
Objective
Methods
Total hip arthroplasty (THA) is increasingly used for active patients with displaced intracapsular hip fractures. Dislocation rates in this cohort remain high postoperatively compared to elective practice, yet it remains unclear which patients are most at risk. The aim of this study was to determine the dislocation rate for these patients and to evaluate the contributing patient and surgeon factors. A five-year retrospective analysis of all patients receiving THA for displaced intracapsular hip fractures from 2013–18 was performed. Data was collected from the institutions' hip fracture database, including data submitted to the National Hip Fracture Database (NHFD). Cox regression analysis and log-rank tests were implemented to evaluate factors associated with THA dislocation. Patient age, sex, ASA grade, surgeon seniority, surgical approach, femoral head diameter and acetabular cup type were all investigated as independent factors.Background
Methods
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.
Massive rotator cuff repairs have up to 60% failure rate and repair of a chronic repair can have up to 40% failure rate. With this in mind, new methodologies are being to being developed to overcome this problem. The use of tendon augmentation grafts is one of them. Prior attempts have shown equivocal or poorer outcomes to control repairs. Aims and objectives: The specific aim of these expereiments was to test how well ovine tendon cells would take to a specific biological augmentation graft (Ligamimetic), and wheter tissue engineering techniques would enhance this. Tendon cells harvested from ovine tendons will be cultured, exposed to the tendon augmentation graft, and analysed to see how well it takes to the tendon cells. We have conducted a 21 day experiment, sampling at days 7, 14, and 21. The experiment will look in sheep tendon cells:1. Platelet rich plasma: A comparison of the effects of platelet rich plasma to cell adherence, cell proliferation, and collagen production. Mesenchymal stem cell: A comparison of the effects of mesenchymal stem cells to the material on cell adherence, cell proliferation, and collagen production.Introduction
Method
Rotator cuff tears remain a problem, with massive tears having a failure rate of repair reported of up to 60%, despite advances in surgical techniques. Tissue engineering techniques offers the possibility of regenerating damaged tendon tissue to a pre-injury state. We explore these techniques by implanting two novel tendon augmentation grafts with use of platelet rich plasma (PRP) in sheep. A total of 24 sheep were operated on, with the infraspinatus being surgically cut from its attachment to the humeral head. Each tendon was repaired using suture anchors and an interpositional implant according to 4 groups: (1) Empty control, (2) Novel collagen fibre implant with PRP (3) A novel collagen sponge implant (4) and the collagen sponge with PRP. The sheep were killed at 12 weeks and the implant site harvested and its histology evaluated.Introduction
Methods
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 inclusion of mineral substantially improves the osteoconductivity of Col/GAG. No significant difference between the different calcium phosphates was seen. Whilst these materials did not stimulate bone formation to the same extent as autograft, many bone graft procedures are carried out with allograft which performs less favourably.Conclusions
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). MBG was prepared using femoral heads donated from patients undergoing total hip replacement surgery using a bone mill in a standard manner. Non porous HA (npHA) was prepared using a precipitation method of Calcium Carbonate and Orthophosphoric acid. The porous HA, (pHA) is a 60% macroporosity HA commercially prepared. The uniaxial compression chamber was a 30mm diameter, steel chamber. Holes were drilled to allow fluid drainage. Loads were applied using a 10 kN load cell. Specimens were prepared in the volumetric proportions pure HA, pure MBG, 2:1, 1:1, 1:2 ratio of MBG to HA. The samples were subjected to compressive forces of incrementally increasing loads of up to 2 KN for 60 cycles. The sample was then allowed to creep under a stress of 2 kN. MBG was also tested up to forces of 7 kN. The mechanical parameters that were examined were the stiffness of the sample at the 60th cycle, (Ec60), and creepMaterials and methods
Chamber
The purpose of this study was to investigate whether combining PRP or concentrated bone marrow aspirate (CBMA) with a biphasic collagen/glycosaminoglycan (CG) scaffold would improve the outcome of the treatment of full thickness osteochondral defects in sheep. Osteochondral defects (5.8×6mm) were created in the medial femoral condyle (MFC) and the lateral trochlea sulcus (LTS) of the stifle joints of 24 sheep. Defects were either left empty or filled with a 6×6mm CG scaffold, either on its own or in combination with PRP or CBMA (n=6). At 6 months the sheep were euthanised, and the repair tissue subjected to mechanical testing, gross morphological analysis, semi quantitative histological scoring and immunohistochemical staining including types I, II and VI collagen.Introduction
Materials and Methods
The aim of this study was to show the efficacy of Chondromimetic in repairing a surgically created osteochondral defect in a caprine model.
The aim of this study was to ascertain whether the addition of thrombin is required to achieve platelet activation and sustained growth factor release in-vitro, when PRP is applied to a collagen based osteochondral scaffold.
Variability in femoral head preparation and high cement pressures may be associated with failure to seat femoral components during hip resurfacing. Furthermore, excessive pressures may lead to over penetration of bone by cement with resulting necrosis of the underlying bone. We designed an experimental model to test the hypothesis that partial-length pressure-relief slots made longitudinally in the proximal bone of the femoral head, without extending to the head neck junction, would allow controlled leakage of cement during initial insertion of a femoral head resurfacing component, but would then become sealed during final insertion to prevent excessive loss of cement while still allowing accurate seating of the component. Thirty-one resurfacing femoral components were cemented onto foam femoral head models. The clearance between foam model and implant was measured to determine the minimum space available for cement. Eleven components were inserted using hand pressure alone, 20 were hammered. Pressure relief slots were prepared in 10 femoral heads. The slots, 4mm deep grooves, were made in the proximal bone only, without extending to the head-neck junction. Cement pressure inside the component was measured during insertion. Implants were sectioned after implantation in order to determine whether they had been fully seated or not. The clinical relevance of the measures taken was tested by measuring the diameter of prepared femoral heads during 20 hip resurfacing operations in order to determine the extent of variability in intra-operative femoral head preparation. Mean intraoperative clearance between bone and implant was −0.19mm (0.11 to −0.93mm). Mean clearance between foam model and implant was −0.30mm (0.35 to −0.94mm). Full seating was obtained in 22/31 components. Of those not fully seated, all had clearance less than −0.74mm. Full seating with a clearance of less than −0.35mm was only possible when pressure relief slots had been made in the femur. The use of a pressure relief slot longer than half the femoral head length allowed full seating in 9/9 cases, compared to 13/22 without. Cement pressure obtained with a hand pressure technique was less than half that observed with hammering (20.8vs56.0psi, p=0.0009) but was not associated with failure to seat the implant if a slot was used. Variability of the actual diameter of the femoral head prepared may be associated with difficulty in fully seating resurfacing components. The same degree of variability in the space available for cement was observed in both intra-operative and test specimens. The use of a pressure-relief slot allows full seating of resurfacing implants with hand pressure alone, thereby halving cement pressure, in an experimental model, even when clearance between implant and bone is less than optimal.
Articular cartilage repair remains a challenge to surgeons and basic scientists. The field of tissue engineering allows the simultaneous use of material scaffolds, cells and signalling molecules to attempt to modulate the regenerative tissue. This review summarises the research that has been undertaken to date using this approach, with a particular emphasis on those techniques that have been introduced into clinical practice, via in vitro and preclinical studies.
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.
This study investigated the effects of wear particles, produced from a number of implant materials, at the bone-implant interface using a small animal model. Particles were prepared from metal, ceramic and polymer replacement joint components or implant grade stock by grinding the materials against a diamond embedded grinding pad. The mean diameter of the particles ranged from 1.5mm to 3.2mm. Sterilised particles were suspended in sterile saline containing 2% v/v male Sprague-Dawley serum at a concentration of 109 particles per ml. Seventy-two male Sprague-Dawley rats were assigned to twelve groups of six animals. A ceramic pin was inserted into the right tibia of each animal. Six groups were assigned a particle type with one group acting as vehicle control. 100ml of particle suspension or vehicle was injected into each knee joint at 8, 10 and 12 weeks following implantation and the animals were killed 2 weeks later. Of the remaining five groups, four were assigned a particle type and one was the vehicle control. These animals were injected with 100ml of particle suspension or vehicle at 20, 22 and 24 weeks following pin implantation and were killed 2 weeks later. The tibia and femora were removed, disarticulated and processed for histology. The total gap between pin and bone, including fibrous tissue, was measured. Specimens showed no signs of infection either clinically or in the histopathology. All materials tested produced lesions at the bone-implant interface. A significant difference was seen between metal injected vs. vehicle control animals and aluminium oxide injected vs. vehicle controls. Particles of stainless steel produced the greatest response and this finding may have implications for the use of metal on metal articulations aimed at eliminating polyethylene wear.
This study investigated the relationship between histological, clinical and radiological features of aseptically loose total joint replacements (TJRs) and synovial fluid levels of interleukin (IL)-1b, IL-6, IL-8 and IL-10. Tissue and synovial fluid samples were retrieved from patients undergoing primary (hip; n=15: knee; n=13), or revision of aseptically loose TJRs (hip; n=14: knee; n=9). The presence of inflammatory cells, blood vessels and wear debris in the tissue were assessed on a relative scale. Revision TJRs were assessed for sepsis, migration of the implant, gross loosening and the degree of radiolucency. Cytokine levels in the synovial fluid samples were determined by ELISA. All cytokines were increased in synovial fluid from revision TJRs compared to primary replacements, as were the degree of macrophage and giant cell infiltration (p<
0.01). There was a significant positive correlation between the presence of macrophages and giant cells with the levels of IL-1b, IL-8 and IL-10 (p<
0.05) but not IL-6. The amount of wear debris was related to the presence of macrophages and giant cells (p<
0.01) but not to any of the cytokines. There were no relationships between any of the clinical parameters and the presence of wear debris or the levels of any cytokine with the exception of IL-6 and gross loosening (p<
0.01). Similarly there were no differences between hips and knees for any of the parameters except IL-6, for which higher levels were found in hips (p<
0.05). The results suggest that macrophages and giant cells are responsible for the majority of IL-1b, IL-8 and IL-10 production but another cell type is contributing to IL-6 production. Furthermore, IL-6 does not fit the pattern of the other cytokines as it is upregulated in hip joints compared to knees and correlates with the presence of gross loosening. This may suggest a unique role for IL-6 that requires further investigation.