INTRODUCTION

Fresh bipolar shell osteochondral allograft (FBOA) is a controversial treatment option for post-traumatic ankle arthritis. Immunological response to transplanted cartilage may play a role in failure. Aim of the study is to compare two groups of patients who received FBOA in association or not to immunosuppressive therapy.

Background

Stem cell based intervertebral disc (IVD) regeneration is quickly moving towards clinical applications. However, many aspects need to be investigated to routinely translate this therapy to clinical applications, in particular, the most efficient way to deliver cell to the IVD. Cells are commonly delivered to the IVD through the annulus fibrosus (AF) injection. However, recent studies have shown serious drawbacks of this approach. As an alternative we have described and tested a new surgical approach to the IVD via the endplate-pedicles (transpedicular approach). The Purpose of the study was to test MSCs/hydrogel transplantation for IVD regeneration in a grade IV preclinical model of IDD on large size animals via the transpeducular approach with cell dose escalation.

Introduction: Following osteochondral transplantation for articular surface defects, union between graft and recipient bed cartilage may occur via two mechanisms. Healing could occur as a result of ingrowths of mesen-chymal cells derived from the subchondral bone. Direct cartilage-to-cartilage healing could occur as a result of chondrocyte proliferation and migration from the margins of graft and recipient bed. This latter mechanism depends upon the marginal chondrocytes surviving the transplantation process, remaining viable and then being capable of cell division as well as normal matrix production.

Aim: The purpose of this study was to investigate the viability of chondrocytes at the graft-recipient bed boundary using the Trypan Blue exclusion technique.

Method: Under general anaesthesia, the medial femoral condyle (MFC) of the right knee of 12 adult male New Zealand White rabbits was exposed via a midline incision and medial arthrotomy. A cylindrical 4mm diameter and 4mm long osteochondral graft was obtained using the T- handle harvester (MITEK COR System) and then it was reinserted into the same site. A groin-to-toe plaster of Paris cast was applied and the animals were allowed to recover. At weekly intervals, 3 animals were killed and the femoral artery of the operated leg was perfused with 10ml Trypan blue. The MFC was excised and fixed in 10% buffered formalin for 1 week. Thereafter, the specimens were decalcified in 10% Kristensen’s solution for 1 week, processed and then paraffin embedded. Sections ‘6u thick were obtained and examined with a light microscope. For each specimen, one section was counterstained with eosin before microscopy.

Results: The animals survived for the duration of the study and the wounds were well healed with no signs of infection. Joint effusion and synovitis were observed in the operated knees at weeks 1, 2 and 3. All grafts were in place and all had faint demarcating borders separating the graft from the surrounding recipient bed. In all cases, there was a zone of positively staining chondrocytes on the periphery of the graft and in the adjoining recipient bed. The zone of positively staining cells extended some considerable distance into the cartilage and affected all its layers. Chondrocytes at the periphery of osteochondral grafts and the adjoining recipient bed may not survive transplantation.

Discussion: This calls into question the ability to achieve direct union between the graft and the recipient bed cartilages. The likely causes of cell death are physical perturbation and direct contact between chondrocytes and blood or synovial fluid. The long-term survival of an osteochondral graft may be determined by whether or not boundary healing has occurred. In the absence of boundary healing, a graft could become bathed in synovial fluid. A pseudarthrosis of sorts could then form which may erode the graft, cause graft subsidence and/or ultimately result in graft death.

Purpose: Osteochondral grafts are being commonly used to repair articular surface defects. The purpose is to achieve the normal architecture of hyaline cartilage with secure and seamless incorporation into recipient sites. However the details of the incorporation of these grafts have not yet been completely elucidated. The expectation is that graft union would involve the proliferation and/or migration of cells and the secretion of matrix and fibres into the graft-host cleft. The aim of this study was to determine the composition of the graft-to-host repair tissue and the integrity of the surfaces of the transplanted graft.

Method: The medial femoral condyle (WC) of the right knee of 12 adult male New Zealand White rabbits was exposed via a midline incision and medial arthrotomy under a general anaesthetic. A cylindrical 4mm diameter and 4mm long osteochondral graft was obtained using the T- handle harvester (MITEK COR System) and then it was reinserted into the same site. A groin-to-toe plaster of Paris cast was applied and the animals were allowed to recover. At weekly intervals, 3 animals were killed and the MFC was excised, fixed in 10% buffered formalin for a week and decalcified in Kristensen’s solution for another week. The specimens were dehydrated through graded ethanol and amyl acetate. Next, they were critical point dried in Blazers Critical Point Drier CPD 030 giving four 15-minute exchanges through liquid C02 before critical point drying. Finally, the specimens were mounted on aluminium stubs and sputter coated in a Polaron SC7640 Sputter coater for 90 seconds resulting in a layer of Gold/Palladium with an approximate thickness of 673 Ao. The samples were then viewed in the Hitachi S-300H scanning electron microscope.

Results: Cartilage-to-cartilage union was not observed at any time interval. Where cartilage union appeared to have occurred, this was due primarily to press fit or ‘surface weld’. In some cases, the adjoining graft and host surfaces revealed superficial fractures presumably caused, as grafts were malleted into place. There was bony union at the base in all cases. In the later time intervals this union had crept up towards the joint surface. The materials in the cleft between the graft and the recipient bed ranged from fibrous to bony elements. The graft surfaces were smooth like the surrounding normal articular cartilage at 1 and 2 weeks but fibrillated at 3 and 4 weeks.

Conclusions: These results appear to suggest that direct cartilage-to-cartilage healing may not occur following osteochondral grafting. Bone-to-bone healing appears to be universal and rapid and, materials ftom this source may be responsible for gap healing. The results also raise the possibility that the articular surfaces of grafts may deteriorate with time but the reasons are not apparent from this study.

1. Homografts of eighteen-day-old foetal femora in pure strains of mice showed no fundamental difference in behaviour from grafts of more mature bone and cartilage.

2. Growth of bone was limited to a short period after transplantation and was abolished by previous immunisation. Cartilage growth alone was responsible for the increase in size of these transplants and did not appear to be influenced by the presence of immunity.

3. There is no reason to suppose that bone from an immature source is likely to behave more favourably than more mature bone homografts in clinical use.

4. The limited growth of cartilage and the total failure of bone survival in the heterografts indicate an immune reaction ofa different order from that which develops against the homograft.

5. The different effect of the homograft immune reaction on cartilage and bone enabled certain conclusions to be drawn concerning the part played by these two tissues in determining the form of a bone. Cartilage growth and development is shown to be regulated in large part by intrinsic factors. Bone growth and form on the other hand is shown to be dependent largely upon extrinsic influences.

Background: A sharp, localised, thoracolumbar gibbus is pathognomonic of the mucopolysaccharidosis (MPS) group of disorders, the most common of which is Hurlers syndrome (MPS I). Untreated patients with this disease run an inevitable course of neurological and physical degeneration until death within the first decade. Haemopoietic stem cell transplantation (HSCT) has resulted in considerable improvement in survival with amelioration of many of the symptoms and signs which characterise this disease. Data, however, is disappointing in relation to the impact of HSCT on skeletal dysplasia. This study reviews the natural history of spinal deformity in Hurler’s syndrome after HSCT in infancy.

Methods: Twenty three patients (12 male and 11 female), transplanted at a mean age of 0.9 years ± 0.47, (range 0.27 – 1.8yrs) were investigated, of whom 19 were at least two years post-HSCT and were included. HLA identical donor sources included unaffected or heterozygote family members, unrelated adults or cord blood. Mean age at review was 9.4 years ± 4.57, (range 2.5 – 18.4yrs). Serial measurements of the thoracolumbar spines incorporated clinical records, radiographs and surface topography. The thoracolumbar gibbus was measured on lateral spinal radiograph using the standard adaptation of the Cobb method. Two segments of the spine were documented: the gibbus itself and the thoracic profile above it. Clinical assessment and surface topography were contrasted with this.

Results: At presentation, all showed the characteristic gibbus at the thoracolumbar junction, with a flat and stiff thoracic spine above. Three patients underwent surgery to correct or maintain the gibbus, which was unsuccessful in two; the third is stable, but still young. Two patients have developed scoliosis: one in the juvenile period and one in infancy. Three female patients are now post-menarchal and have shown no progression of their gibbus. One male patient, now aged 19 years, had significant progression of his gibbus at puberty, but is now stable, untreated and cosmetically acceptable. The remainder are still pre-pubertal but their deformities are not currently progressive.

Conclusion: The fate of the spinal deformity in untreated MPS-I has been poorly documented, as the condition was invariably fatal from cardiorespiratory failure during the first decade. These interim results suggest that, while the deformity persists and may become more pronounced during growth and adolescence, it does not significantly impact on quality of life. The considerations which usually dictate intervention in other spinal deformities of childhood may not necessarily apply and should be approached with caution. The more recent availability of recombinant human -L- iduronidase adds further interest to the management of these patients and warrants cautious expectation , in the context of experience gained in these groups of patients. In conclusion atients with MPS I have complex multisystem disorders, independent of their orthopaedic status. While monitoring their spinal deformity is indicated, over-intrusive investigation and treatment may be counterproductive.

Background: Autologous mesenchymal stem cells (MSC) have been shown to improve the functional outcome after severe skeletal muscle trauma. The reasons for this improvement have yet not been revealed. Up to now insufficient techniques of cell labelling, which could only be used for histologic analysis ex vivo, have been a problem.

The development of iron oxide nanoparticles, which are taken up and endosomally stored by stem cells, allows the evaluation of cellular behaviour in the muscle with the use of magnetic resonance imaging (MRI). Previous work has shown that labelling does not affect the proliferation and neurogenic differentiation capacity of embryonic stem cells. In the present study we are currently investigating the in vivo distribution and migration of locally transplanted MSC after blunt muscle trauma in a rat model.

Methods: MSC cultures are derived from tibial biopsies of Sprague Dawley rats via plastic adherence. A standardized open crush injury of the left soleus muscle is performed in each animal. 24 hours before transplantation cells are labelled with very small superparamagnetic iron oxid particles (VSOP-C200, Ferropharm, Teltow, Germany) and Green Fluorescent Protein (GFP). One week after trauma different amounts of stem cells (5×105, 1×106 and 5×106) are transplanted into the soleus muscle by local injection. Distribution and migration of the cells are evaluated over time by the repeated performance of high resolution-MRI at 7 Tesla (Bruker, Rheinstetten, Germany). At the endpoint of the study, three and six weeks after transplantation, the muscles are harvested and histologically and immunohistochemically analysed.

Results: Cells could be visualised inside the soleus muscle in the MRI 24 hours after transplantation showing characteristic signal extinctions in T2*-weighed images. The hypointense signal could be followed over the longest investigated time of six weeks and could be easily discriminated from the structures of the injured muscle. Preliminary results show that the cell pool changed its shape over time with the loss of an initially depicted injection canal and an increase in the surface/volume ratio. First histologic Prussian Blue stained sections showed co-localisation of the respective MRI signal and nanoparticle labelled cells. Fusion events of marked cells with regenerating myofibers could be observed.

Conclusion: Magnetic labelling of MSC is a powerful tool to analyse the in vivo behaviour of the cells after transplantation into a severly injured skeletal muscle. For the first time the observation of an intraindividual time course of the distribution of the transplanted cells is possible. Our preliminary results are promising and the ongoing work will further characterise migration processes and the correlation of the MRI results with muscle function evaluated by contraction force measurements.

Mesenchymal stem cells (MSCs) from bone marrow are multipotent cells capable of forming cartilage, bone, and other connective tissues. The objective of this study was to determine whether the use of allogenic mesenchymal stem cells could functionally heal defect in the distal femoral physis in rabbits without the use of immunosuppressive therapy. An iatrogenic defect was created in the lateral femoral condyle of thirty-two New Zealand white rabbits, 7 weeks old, that weighed 2.25 ? 0.24 kg. Each defect, 3.5 mm in width and 12 mm in length, in the right distal femoral physis was treated with allogenic mesenchymal stem cells in new composite hyaluronate/ collagen type I/fibrin scaffold. The healing response was evaluated radiographically, by MRI (at three weeks and four months after implantation), and also histologically, by Pearls’ reaction and with immunofluorescency (at four months after implantation). The results were compared with the data for the control defects (without stem cell implantation) in left distal femoral physes. In average, right femurs with damaged distal physis and transplanted MSCs grew more in length (0.55? 0.21 cm) as compared with left femurs with physeal defect without stem cell transplantation (0.46? 0.23 cm). Valgus deformity of right femurs with physeal defect and transplanted MSCs was mild (0.2? 0.1°). On the contrary, left femurs with physeal defect without transplantated MSCs showed significant valgus deformity (2.7? 1.6°). For defects treated with allogenic mesenchymal stem cell implants, no adverse immune response and implant rejection were detected in this model. Histologically, no lymphocytic infiltration occurred. At four months after transplantation, hyalinne cartilage had formed throughout the defects treated with allogenic MSCs. Labeled mesenchymal stem cells/diferentiated chondrocytes were detected in the physeal defects based on magnetic resonance imaging and immunofluorescency. The results of this study demonstrated that allogenic mesenchymal stem cells in a new composite hyaluronate/collagen type I/fibrin scaffold repaired iatrogenic defects in the distal femoral physes in rabbits without the use of immunosuppressive therapy. The use of allogenic mesenchymal stem cells for the repair of physeal defects may be an alternative to autologous MSCs transplantation. An allogenic approach would enable mesenchymal stem cells to be isolated from any donor, providing a readily available source of cells for cartilage tissue repair.

For the treatment of ununited fractures, we developed a system of delivering magnetic labelled mesenchymal stromal cells (MSCs) using an extracorporeal magnetic device. In this study, we transplanted ferucarbotran-labelled and luciferase-positive bone marrow-derived MSCs into a non-healing femoral fracture rat model in the presence of a magnetic field. The biological fate of the transplanted MSCs was observed using luciferase-based bioluminescence imaging and we found that the number of MSC derived photons increased from day one to day three and thereafter decreased over time. The magnetic cell delivery system induced the accumulation of photons at the fracture site, while also retaining higher photon intensity from day three to week four. Furthermore, radiological and histological findings suggested improved callus formation and endochondral ossification. We therefore believe that this delivery system may be a promising option for bone regeneration.

Introduction Intervertebral disc degeneration may cause chronic low back pain. Disc degeneration is characterized by dysfunctional cells and a decrease in extra-cellular components. Bone marrow derived mononuclear cells are a heterogeneous cell population which contains mesenchymal stem cells. Transplantation of stem cells and progenitor cells may provide a new approach to treat disc degeneration, but it is unclear if transplanted cells can survive and differentiate in the non-vascularized disc. Methods Bone marrow was collected from syngeneic Sprague-Dawley rats and mononuclear cells were isolated. The cells were labelled with a fluorescence dye (Cell Tracker Orange) and suspended in PBS. 10–20μl of the cell suspension (1–2x10. 5. cells/disc) was transplanted into coccygeal discs in 12 syngeneic rats. For each rat two discs were cell transplanted and one disc served as control. The rats were sacrificed after 0, 7, 14 or 21 days. For each time point the discs from one animal were saved for routine histological staining. The cell transplanted discs of the other animals (n=4 discs per time point) were formalin-fixed, frozen and sectioned together with the control discs. Frozen disc sections were visualized with fluorescence microscopy and the number of transplanted cells assessed. Expression of collagen II, a marker of chondrocytes and chondrocyte-like cells in the disc, was assessed in the transplanted cells using immunofluorescence technique. Results All cell-suspension injected discs contained transplanted bone-marrow cells. The discs within each time-group demonstrated a large variation in number of detected cells. There was a decrease in detected cells at 7, 14 and 21 days compared to day 0. Transplanted cells expressed collagen II after 21 days but not after 7 and 14 days. Discussion The results suggest that transplanted bone marrow-derived mononuclear cells can survive and differentiate within the intervertebral disc. Further studies in models of disc degeneration are warranted to investigate the regenerative potential of the disc following cell transplantation

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

Objectives

In order to ensure safety of the cell-based therapy for bone regeneration, we examined in vivo biodistribution of locally or systemically transplanted osteoblast-like cells generated from bone marrow (BM) derived mononuclear cells.