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

Background

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.

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.

Introduction

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.

Introduction

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.

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).

Introduction

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.

Introduction: Platelet rich plasma (PRP) has been hypothesised to be of potential benefit to articular cartilage tissue engineering, through its release of autologous growth factors. 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.

Methods: Collagen/glycosaminoglycan scaffolds were fashioned, to which equal combined volumes of test substances were added (n=3): 500μl PRP; 375μl PRP + 125μl autologous thrombin (3:1); 455μl PRP + 45μl bovine thrombin (10:1). One ml of DMEM/F12 medium was added to each scaffold and changed completely at 12/24 hours, and 3/10 days, following which release of TGF-β1, PDGF-AB and bFGF were measured using ELISA. Secondly, equal sized collagen/glycosaminoglycan and polylactide co-glycolide scaffolds were fashioned to which 500μl of PRP were added (n=3). Similar conditions were followed as previously except that only PDGF-AB was assayed.

Results: A similar cumulative release profile of all growth factors was found over the 10 day period. An increase in growth factor release was seen in the PRP only group at all time points with PDGF-AB in particular reaching statistical significance at all time points (p< 0.006). These findings remained apparent when a correction for volume was made (p< 0.028) suggesting a particular role of the collagen in platelet activation. This was shown in the second experiment, in which a significantly increased cumulative volume of PDGF-AB was released from the collagen/glycosaminoglycan scaffold without thrombin activation (p< 0.04).

Discussion: This study shows that collagen is a potent activator of platelets, requiring no further addition to achieve satisfactory growth factor release when applied clinically. These results suggest that if PRP is combined with polymer scaffolds, it should be activated with thrombin to achieve optimum growth factor release.

Introduction: Current treatment options for small, contained articular cartilage defects include microfracture, osteochondral autograft plugs or newer synthetic plugs. Chondromimetic is a novel biphasic biological scaffold composed of collagen and glycosaminoglycan. The addition of brushite provides the scaffold with a regionally specific component mimicking both phases of the osteochondral unit. The aim of this study was to show the efficacy of Chondromimetic in repairing a surgically created osteochondral defect in a caprine model.

Methods: Osteochondral defects were made in the lateral trochlear sulcus (LTS) and medial femoral condyle (MFC) of nine goats. Chondromimetic scaffolds (6x6mm) were inserted into each defect (n=6), while three controls had defects left empty (n=3). All animals were sacrificed at 26 weeks postoperatively. Macroscopic evaluations and quantitative stiffness properties were assessed. Histological sections were taken at approximately the centre of the defect, stained with Safrinin O/Fast Green and scored using a validated quantitative assessment tool.

Results: Macroscopically, the repair tissue scored higher in the MFC and LTS (p< 0.05) compared to controls. In all defects, the mechanical stiffness was found to be within one standard deviation of native cartilage, except that of the LTS controls. Histologically, the predominant tissue in the cartilage layer was deemed to be hyaline-like in three of six MFC defects, and five of six LTS defects according to the modified Sellers score. This was compared to one in three and zero of three in the MFC and LTS controls respectively.

Discussion: These results represent the early findings from an ongoing in-vivo study in which a further group of animals will be sacrificed at one year. At six months, the histology and mechanical properties are encouraging and should continue to improve with time. These results show that Chondromimetic may represent an acceptable alternative to marrow stimulation in the treatment of osteochondral defects.

Introduction: Platelet rich plasma (PRP) has been hypothesised to be of potential benefit to articular cartilage tissue engineering, through its release of autologous growth factors. 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.

Methods: Collagen/glycosaminoglycan scaffolds were fashioned, to which equal combined volumes of test substances were added (n=3): 500μl PRP; 375μl PRP + 125μl autologous thrombin (3:1); 455μl PRP + 45μl bovine thrombin (10:1). One ml of DMEM/F12 medium was added to each scaffold and changed completely at 12/24 hours, and 3/10 days, following which release of TGF-β1, PDGF-AB and bFGF were measured using ELISA. Secondly, equal sized collagen/glycosaminogly-can and polylactide co-glycolide scaffolds were fashioned to which 500μl of PRP were added (n=3). Similar conditions were followed as previously except that only PDGF-AB was assayed.

Results: A similar cumulative release profile of all growth factors was found over the 10 day period. An increase in growth factor release was seen in the PRP only group at all time points with PDGF-AB in particular reaching statistical significance at all time points (p< 0.006). These findings remained apparent when a correction for volume was made (p< 0.028) suggesting a particular role of the collagen in platelet activation. This was shown in the second experiment, in which a significantly increased cumulative volume of PDGF-AB was released from the collagen/glycosaminoglycan scaffold without thrombin activation (p< 0.04).

Discussion: This study shows that collagen is a potent activator of platelets, requiring no further additive to achieve satisfactory growth factor release when applied clinically. These results suggest that if PRP is combined with polymer scaffolds, it should be activated with thrombin to achieve optimum growth factor release.

Introduction: Current treatment options for small, contained articular cartilage defects include microfracture, osteochondral autograft plugs or newer synthetic plugs. Chondromimetic is a novel biphasic biological scaffold composed of collagen and glycosaminoglycan. The addition of brushite provides the scaffold with a regionally specific component enabling the scaffold to mimic both phases of the osteochondral unit.

The aim of this study was to show the efficacy of Chondromimetic in repairing a surgically created osteochondral defect in a caprine model.

Methods: Osteochondral defects were made in the lateral trochlear sulcus (LTS) and medial femoral condyle (MFC) of nine goats. Chondromimetic scaffolds (6x6mm) were inserted into each defect (n=6), while three controls had defects left empty (n=3). All animals were sacrificed at 26 weeks postoperatively. Macroscopic evaluations and quantitative stiffness properties were assessed. Histological sections were taken at approximately the centre of the defect, stained with Safrinin O/Fast Green and scored using a validated quantitative assessment tool.

Results: Macroscopically, the repair tissue scored higher in the filled MFC and LTS (p< 0.05) compared to controls. In all defects, the mechanical stiffness was found to be within one standard deviation of native cartilage, except the LTS controls. Histologically, the predominant tissue in the cartilage layer was hyaline-like in three of six filled MFC defects, and five of six filled LTS defects according to the modified Sellers score. This was compared to one in three and zero of three in the MFC and LTS controls respectively.

Discussion: These results represent the early findings from an ongoing in-vivo study in which a further group of animals will be sacrificed at one year. At six months, the histology and mechanical properties are encouraging and should continue to improve with time. These results show that Chondromimetic may represent an acceptable alternative to marrow stimulation in the treatment of osteochondral defects.

Introduction: Platelet rich plasma (PRP) has been hypothesised to be of potential benefit to articular cartilage tissue engineering, through its release of autologous growth factors.

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.

Methods: Collagen/glycosaminoglycan scaffolds were fashioned, to which equal combined volumes of test substances were added (n=3): 500μl PRP; 375μl PRP + 125μl autologous thrombin (3:1); 455μl PRP + 45μl bovine thrombin (10:1). One ml of DMEM/F12 medium was added to each scaffold and changed completely at 12/24 hours, and 3/10 days, following which release of TGF-β1, PDGF-AB and bFGF were measured using ELISA. Secondly, equal sized collagen/glycosaminogly-can and polylactide co-glycolide scaffolds were fashioned to which 500μl of PRP were added (n=3). Similar conditions were followed as previously except that only PDGF-AB was assayed.

Results: A similar cumulative release profile of all growth factors was found over the 10 day period. Greater growth factor release was seen in the PRP only group at all time points with PDGF-AB in particular reaching statistical significance at all time points (p< 0.006). These findings remained apparent when a correction for volume was made (p< 0.028) suggesting a particular role of the collagen in platelet activation. This was shown in the second experiment, in which a significantly increased cumulative volume of PDGF-AB was released from the collagen/glycosaminoglycan scaffold without thrombin activation (p< 0.04).

Discussion: This study shows that collagen is a potent activator of platelets, requiring no further addition to achieve satisfactory growth factor release when applied clinically. These results suggest that if PRP is combined with polylactide co-glycolide scaffolds, it should be activated with thrombin to achieve optimum growth factor release.

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.