Aims. Ageing-related incompetence becomes a major hurdle for the clinical translation of adult stem cells in the treatment of osteoarthritis (OA). This study aims to investigate the effect of stepwise preconditioning on cellular behaviours in human mesenchymal stem cells (hMSCs) from ageing patients, and to verify their therapeutic effect in an OA animal model. Methods. Mesenchymal stem cells (MSCs) were isolated from ageing patients and preconditioned with chondrogenic differentiation medium, followed by normal growth medium. Cellular assays including Bromodeoxyuridine / 5-bromo-2'-deoxyuridine (BrdU), quantitative polymerase chain reaction (q-PCR), β-Gal, Rosette forming, and histological staining were compared in the manipulated human mesenchymal stem cells (hM-MSCs) and their controls. The anterior cruciate ligament transection (ACLT) rabbit models were locally injected with two millions, four millions, or eight millions of hM-MSCs or phosphate-buffered saline (PBS). Osteoarthritis Research Society International (OARSI) scoring was performed to measure the pathological changes in the affected joints after staining. Micro-CT analysis was conducted to determine the microstructural changes in subchondral bone. Results. Stepwise preconditioning approach significantly enhanced the proliferation and chondrogenic potential of ageing hMSCs at early passage. Interestingly, remarkably lower immunogenicity and senescence was also found in hM-MSCs. Data from animal studies showed cartilage damage was retarded and subchondral bone remodelling was prevented by the treatment of preconditioned MSCs. The therapeutic effect depended on the number of cells applied to animals, with the best effect observed when treated with eight millions of hM-MSCs. Conclusion. This study demonstrated a reliable and feasible stepwise preconditioning strategy to improve the safety and efficacy of ageing MSCs for the prevention of OA development. Cite this article: Bone Joint Res 2021;10(1):10–21

The April 2012 Research Roundup³⁶⁰ looks at who is capable of being an arthroscopist, bupivacaine, triamcinolone and chondrotoxicity, reducing scarring in injured skeletal muscle, horny Goat Weed and the repair of osseous defects, platelet-derived growth factor and fracture healing, the importance of the reserve zone in a child’s growth plate, coping with advanced arthritis, hydroxyapatite and platelet-rich plasma for bone defects, and calcium phosphate and bone regeneration

Platelet-derived growth factor (PDGF) is known to stimulate osteoblast or osteoprogenitor cell activity. We investigated the effect of locally applied PDGF from poly-. d. ,l-lactide (PDLLA)-coated implants on fracture healing in a rat model. A closed fracture of the right tibia of four-month-old Sprague-Dawley rats (n = 40) was stabilised with implants coated with a biodegradable PDLLA versus implants coated with PDLLA and PDGF. Radiographs were taken throughout the study, and a marker of DNA activity, bromodeoxyuridine (BrdU), was injected before the rats were killed at three, seven and ten days. The radiographs showed consolidation of the callus in the PDGF-treated group compared with the control group at all three time points. In the PDGF-treated group, immunohistochemical staining of BrdU showed that the distribution of proliferating cells in all cellular events was higher after ten days compared with that at three and seven days. These results indicate that local application of PDGF from biodegradable PDLLA-coated implants significantly accelerates fracture healing in experimental animals. Further development may help fracture healing in the clinical situation.

The response of the muscle is critical in determining the functional outcome of limb lengthening. We hypothesised that muscle response would vary with age and therefore studied the response of the muscles during tibial lengthening in ten young and ten mature rabbits. A bromodeoxyuridine technique was used to identify the dividing cells. The young rabbits demonstrated a significantly greater proliferative response to the distraction stimulus than the mature ones. This was particularly pronounced at the myotendinous junction, but was also evident within the muscle belly. Younger muscle adapted better to lengthening, suggesting that in patients in whom a large degree of muscle lengthening is required it may be beneficial to carry out this procedure when they are young, in order to achieve the optimal functional result

Little is known about the increase in length of tendons in postnatal life or of their response to limb lengthening procedures. A study was carried out in ten young and nine adult rabbits in which the tibia was lengthened by 20% at two rates 0.8 mm/day and 1.6 mm/day.

The tendon of the flexor digitorum longus (FDL) muscle showed a significant increase in length in response to lengthening of the tibia. The young rabbits exhibited a significantly higher increase in length in the FDL tendon compared with the adults. There was no difference in the amount of lengthening of the FDL tendon at the different rates. Of the increase in length which occurred, 77% was in the proximal half of the tendon.

This investigation demonstrated that tendons have the ability to lengthen during limb distraction. This occurred to a greater extent in the young who showed a higher proliferative response, suggesting that there may be less need for formal tendon lengthening in young children.

Background: Growth and development of the intervertebral disc and its adjacent vertebrae is regulated via relative levels of cell proliferation, cell death and hypertrophy, and through extracellular matrix synthesis or degradation [. 1. ]. The synthesis of matrix molecules in the growing spine of embryonic rats has been reported in some detail [. 2. ,. 3. ]. In addition, increased levels of apoptotic disc cell death have been described in normal ageing, disc degeneration and in a murine model of disc spondylosis [. 4. ,. 5. ]. However, levels of cell proliferation in the developing spine have not been formally investigated. Methods/Results: BALB/c mice were injected with the thymidine analogue, bromodeoxyuridine (BrdU), at weeks 1–4 postnatally and killed 1 or 24 hours later. The lumbar spines were decalcified and tissue sections immunostained for BrdU-incorporation. The intervertebral disc was fully formed at weeks 1–4, consisting of a notochordal nucleus pulposus, lamellar anulus fibrosus, and cartilaginous endplates between the disc and vertebral growth-plates. BrdU-immunopositivity was most marked in 1 week old mice, particularly in the proliferative zone of the growth-plate and the apophyseal ring. By 4 weeks, few, if any, BrdU-labelled cells were present in the disc, but some positivity remained in the apophyses. There were more paired BrdU-labelled cells at 24 hours than 1 hour post-injection in all regions, indicating likely clonal growth of these cells. Conclusions: Cell proliferation forms an important part of the growth of the vertebrae, but also features in the early postnatal growth of the murine intervertebral disc. An understanding of how proliferation in these cell populations is regulated will help augment repair and regenerative responses in damaged adult discs or scoliosis

Introduction: Prior to skeletal maturity temporary hemiepiphyseal stapling is a treatment method for angular deformities of long bones. The purpose of this study is to investigate the effects of temporary hemiepiphyseal stapling on the bone geometry and histology of physis. Materials & Methods: Proximal medial epipyseal stapling of the right tibia were done in 46 New Zealand rabbits. 23 of them were euthanized at the end of 3 weeks. For the remaining 23 rabbits staples were fixed subperiostally (group A) in 11, and extraperiosteally (group B) in 12 rabbits. After 3 weeks the staples removed and the rabbits were euthanized at the end of 6 weeks. Bromodeoxyuridine used to evaluate cellular activity of the growth plate. Radiographs utilized for bone alignment. Results: The articular surface-diaphysis angle was significantly increased at the end three weeks when compared to controls (27.7° vs. −1.5°, p:0.001). Cellular activity was decreased but preserved in the stapled tibias. At the end of six weeks while the angular deformity was worsening in group A 22.9° vs. 35.6°, p:0.001) it was improving in group B (23.2 ° vs. 14.6°, p:0.001). Bone tissue bridging the growth plate was noted in group A. Cellular activity in the group B was higher than group A at the end of six weeks. Conclusion: Hemiepiphyseal stapling causes decreased cellular activity at the growth plate, which leads to angulation. With removal of staples, increased cellular activity at the growth plate results in the improvement of the deformity if staples were inserted extraperiosteally. Temporary extraperiosteal hemiepiphyseal stapling could be used as a safe and effective method for treatment of angular deformities prior to skeletal maturity

Introduction: Eighty percent of individuals experience low back pain in their lifetime. This is often due to disc injury or degeneration. Conservative treatment of discogenic pain is often unsuccessful whilst surgery with the use of spacers or fusion is non-physiological. Aim: To develop an animal model to assess the viability of autologous disc cell therapy. Methods: The fat sand rat (Psammomys obesus obesus) was chosen because of its predisposition to the early development of spondylosis. Using microsurgical techniques fragments of annulus and nucleus were harvested from a single disc in 50 sand rats. Vascular clips were placed on the adjacent psoas muscle to mark the harvested level. Disc material was initially cultured in a monolayer then transferred into a three-dimensional culture medium of agarose. This technique yields greater cellular proliferation and the development of cell growth in colonies. Cells were labelled with bromodeoxyuridine for later immunohistochemical identification. Twenty thousand cells in a carrier medium were then reimplanted at a second operation at an adjacent disc level in the same animal. The rat was subsequently sacrificed and the histology of the disc space was reviewed. Results: To date, 50 primary disc harvests and 30 reimplantations have been performed. Two rats died prior to reimplantation. All histological specimens confirmed the presence of viable transplanted disc cells. Conclusions: Autologous disc cell transplantation can be performed in the rat. Further modification of these techniques may lead to the development of autologous disc cell therapy comparable to that currently successfully used in hyaline cartilage defects of synovial joints in humans

Purpose/introduction: 80% of individuals experience low back pain in their lifetime. This is often due to disc injury or degeneration. Conservative treatment of discogenic pain is often unsuccessful whilst surgery with the use of spacers of fusion is non-physiological. The aim of this study was to develop an animal model to assess the viability of autologous disc cell therapy. Method: The Fat Sand Rat (Psammomys obesus obesus) was chosen due to its predisposition to the early development of spondylosis. Using microsurgical techniques fragments of annulus and nucleus were harvested from a single disc in 52 sand rats. Vascular clips were placed on the adjacent psoas muscle to mark the harvested level. Disc material was initially cultured in monolayer then transferred into a three dimensional culture media of agarose. This technique yields greater cellular proliferation and the development of cell growth in colonies. Cells were labelled with Bromodeoxyuridine for later immunohistochemical identification. 20 000 cells in a carrier media were then re-implanted at a second operation at an adjacent disc level in the same animal. The rat was subsequently euthanised and the histology of the disc space reviewed. Results: To date 52 primary disc harvests and 20 reimplantations have been performed. 15 rats have been euthanised and sectioned. Average age at primary surgery was 6.8 months reimplantation eight months and euthanisation 11.2 months. Cell colony viability was inversely related to rat age at harvest. Immunohistochemical analysis of colony extracellular matrix revealed production of type 1 and 2 collagen, chondroitin and keratin sulphate Two rats died prior to reimplantation. All histological specimens confirm the presence of viable transplanted disc cells. Transplanted cells did not alter the progression of degenerative changes on x-ray. Conclusion: Autologous disc cell transplantation can be performed in the rat. Further modification of these techniques may lead to the development of autologous disc cell therapy comparable to that currently successfully used in hyaline cartilage defects of synovial joints in humans

We studied the cellular response to physeal distraction in the growth plates of skeletally immature rabbits. We used a new method of labelling and detection of proliferating cells with bromodeoxyuridine (BUdR) and an anti-BUdR antibody. The application of an external fixator but no distraction force produced no changes in the growth plates. After five days of distraction at a maximum force of 20 N, the growth plate became thicker, mainly because of an increase in the number of hypertrophic chondrocytes, but there was no evidence of increased cell proliferation. Recent fractures were seen at the junction of growth plate and metaphysis but the increase in bone length was insignificant. After ten days of distraction at the same maximum force, the chondrocyte columns had become disorganised and cell proliferation was significantly decreased. There was an increase in bone length due to distraction of the fracture gap. In this model, physeal distraction did not stimulate cell proliferation, but actually inhibited it. The apparent increase in growth-plate thickness produced by distraction is not due to increased cell production, but results from inhibition of endochondral ossification and the consequent accumulation of hypertrophic chondrocytes. Any growth after distraction depends on the ability of growth-plate chondrocytes to divide. The decrease in proliferative activity which we found after ten days of distraction suggests the need for caution in the use of such procedures in young children