Background and aim: Mesenchymal stem cells (MSCs) are multipotent cells capable of differentiation into osteogenic and chondrogenic pathways. MSCs are among the key repair cells in fracture healing and implant osseointegration. They are also an attractive tool of cell therapy in reconstruction procedures of bone. Minipigs are a large-animal model recommended for preclinical studies of orthopaedic bone implants. Minipigs are claimed to have bone physiology close to humans, but their MSC characteristics are poorly defined. The aim of this study was to isolate and characterize minipig bone marrow and peripheral blood derived MSCs in comparison of human MSCs. Methods: Five male minipigs (weight 36.2 ± 2.2 kg) were subjected for experimental femoral osteotomy, which was fixed with either compression plate or intra-medullary nailing. Before surgery, bone marrow (BM) sample (2–4 ml) was aspirated from the posterior iliac crest and a peripheral blood (PB) sample (20 ml) was also collected. Mononuclear cells (MNC) were isolated by Ficoll gradient centrifugation. MSCs were cultured and selected by plastic adherence. Cell morphology was evaluated during the whole culture period and proliferation capacity was examined by determining the number of population doublings (PDs) at the end of each passage. Osteoblastic differentiation capacity was investigated by culturing MSCs in the presence of beta-glycerophosphate, dexamethasone and ascorbic acid. The lineage phenotype was studied by alkaline phosphatase and von Kossa staining. Results: MNC were successfully isolated from all BM and PB samples. Plastic adherent cells obtained fibroblast-like morphology and proliferated over time in culture. The maximum PDs were 3.4 ± 0.7 and 4.3 ± 0.5 for BM- and PB-derived cells, respectively. The maximum PD capacity of PB-derived cells was significantly higher than that of BM-derived cells (p=0.027). However, when cultured in osteoblastic induction medium, only BM-derived cells were capable of differentiating into alkaline phosphatase positive osteoblasts with an occasional presence of von Kossa-stained mineralized bone nodules. The maximum PDs of minipig BM-derived MSCs were similar to those of human BM-MSCs isolated from young adult fracture patients. Conclusion: We successfully isolated plastic adherent MSCs from minipig bone marrow samples, which proliferated and differentiated into cells of osteoblastic lineage. BM-derived porcine MSCs had similar morphology to human MSCs. There were marked inter-individual variations in the proliferation and differentiation capacity of minipig MSCs, resembling the observations in humans. No circulating MSCs could be detected in minipigs before surgery and this confirmed our previous observation in humans

This study evaluated the effect of a collagen type I /hyaluronate (c/h) implant combined with recombinant human growth and differentiation factor-5 (rhGDF-5) in osteochondral cartilage defects of Göttinger minipigs. In 20 Göttinger minipigs, critical size defects (6.2mm wide and 10mm deep) were created in the medial condyle of both femora. Defects were treated on one side either with the c/h implant alone (n=10) or the c/h implant + rhGDF-5 (n=10), whereas the other side was left empty as an intra-individual control. After 3 and 12 months, 5 animals from each treatment group were killed. The evaluation included macroscopic investigation, biomechanical exploration by relaxation test and semi-quantitative histological scoring using the O’Driscoll score. No macroscopic differences were found between the two treatment groups, neither could any differences be found in semi-quantitative histological scoring. Biomechanical measurement after 12 months showed a significant increase in peak stress in the c/h group compared to empty defects, however, rhGDF-5 supplementation was not found to influence the biomechanical properties compared to controls. Bony cysts were seen throughout the three treatment groups, indicating insufficient bone regeneration. In two animals treated with rhGDF-5, pronounced ossifications within the joint capsule were observed. In contrast, no ossifications were detected in the knees with empty defects or single treatment with c/h implant. In conclusion, the combination of a c/h implant plus rhGDF-5 did not result in better defect regeneration compared to c/h implants alone or even to empty defects in our minipig model. One major problem seems to be the incomplete regeneration of the bony defect when using this device. In further studies, bilayer matrices should be used to address this problem. Due to the small number of specimens in this study, it cannot be resolved whether the ossifications seen in two knees were due to the usage of rhGDF-5 or can be regarded as an independent event. Further data about growth factor interaction should be acquired in animal studies before clinical introduction can be considered

Aim. To make an inoculum for induction of Implant-Associated Osteomyelitis (IAO) in pigs based on bacterial aggregates resembling those found on the human skin, i.e. aggregates of 5–15 µm with low metabolic activity. The aggregates were evaluated and compared to a standard planktonic bacterial inoculum. Method. The porcine Staphylococcus aureus strain S54F9 was cultured in Tryptone Soya Broth for seven days. Subsequently, the culture was filtered through cell strainers with pore sizes of 15 µm and 5 µm, respectively. The fraction of 5–15 µm aggregates in the top of the 5 µm filter was collected as the aggregate-inoculum. The separation of aggregates into different size fractions was evaluated by light microscopy. The metabolism of the aggregate-inoculum and a standard overnight planktonic inoculum was evaluated with isothermal microcalorimetry. In total, six female minipigs were allocated into three groups (n=2), receiving different inoculums. Group A: overnight planktonic inoculum; 10. 4. CFU S. aureus (S54F9), Group B: seven days old 5–15 µm aggregate-inoculum; 10. 4. CFU S. aureus (S54F9), Group C: saline. All inoculums were placed in a pre-drilled implant cavity in the right tibia of the pig and a sterile stainless-steel implant was inserted. The pigs were euthanized seven days after surgery. Postmortem macroscopic pathology, microbiology, computed tomography and histopathology were performed. Results. The separation of aggregates into different size fractions was done successfully by the filtering method. Isothermal microcalorimetry showed, a delayed Time-to-peak metabolic activity of the aggregate-inoculum compared to the planktonic inoculum. S. aureus was isolated from subcutis, bone and implants from all animals in groups A and B. Both group A animals showed osteomyelitis at gross inspection with suppuration and sequestration, while groups B and C animals had no macroscopic lesions. From CT scans, both group A animals also showed positive signs of osteomyelitis, i.e., osteolysis, while only one animal in group B did, and none in group C. Histopathological examination of the bones showed more extensive inflammation in group A animals compared to those in group B, which showed more osteoid formation. Conclusions. Formation and separation of low metabolism bacterial aggregates into different size fractions was possible. The aggregates can be used as inoculum in the porcine IAO model, with microbiological re-isolation from both implants and tissue. Furthermore, the aggregates caused a less aggressive IAO, than the planktonic counterparts. Using aggregated bacteria as inoculum appears to be more relevant to the clinical situation of infecting bacteria

Long-term regeneration of cartilage defects treated with tissue engineering constructs often fails because of insufficient integration with the host tissue. We hypothesize that construct integration will be improved when implants actively interact with and integrate into the subchondral bone. Growth and Differentiation Factor 5 (GDF-5) is known to support maturation of chondrocytes and to enhance chondrogenic differentiation and hypertrophy of mesenchymal stromal cells (MSC). Therefore, we investigated whether GDF-5 is capable to stimulate endochondral ossification of MSC in vitro and in vivo and would, thus, be a promising candidate for augmenting fibrin glue in order to support integration of tissue engineering constructs into the subchondral bone plate. To evaluate the adhesive strength of fibrin glue versus BioGlue. ®. , a commercially available glue used in vascular surgery, an ex vivo cadaver study was performed and adhesion strength was measured via pull-out testing. MSC were suspended in fibrin glue and cultivated in chondrogenic medium with and without 150 ng/mL GDF-5. After 4 weeks, the formed cartilage was evaluated and half of the constructs were implanted subcutaneously into immunodeficient mice. Endochondral ossification was evaluated after 2 and 4 weeks histologically and by microCT analysis. BioGlue. ®. and GDF-5-augmented fibrin glue were tested for 4 weeks in a minipig cartilage defect model to assess their orthotopic biocompatibility. Pull-out testing revealed sufficient adhesive strength of fibrin glue to fix polymeric CellCoTec constructs in 6 mm cartilage defects, however, BioGlue. ®. showed significantly higher adhesive power. In vitro chondrogenesis of MSC under GDF-5 treatment resulted in equal GAG deposition and COLIIa1 and ACAN gene expression compared to controls. Importantly, significantly increased ALP-activity under treatment with GDF-5 on day 28 indicated enhanced hypertrophic differentiation compared to controls. In vivo, MSC-fibrin constructs pre-cultured with GDF-5 developed a significantly higher bone volume on day 14 and 28 compared to controls. When pre-cultured with GDF-5 constructs showed furthermore a significantly higher bone compactness (bone surface/bone volume coefficient) than controls, and thus revealed a higher maturity of the formed bone at 2 weeks and 4 weeks. Orthotopic biocompatibility testing in minipigs showed good defect filling and no adverse reactions of the subchondral bone plate for defects treated with GDF-5-augmented fibrin glue. Defects treated with BioGlue. ®. , however, showed considerable subchondral bone lysis. Thus, BioGlue. ®. – despite its adhesive strength – should not be used for construct fixation in cartilage defects. GDF-5-augmented fibrin glue is considered promising, because of a combination of the adhesive strength of fibrin with an enhanced osteochondral activity of GDF-5 on MSC. Next step is to perform a large animal study to unravel whether GDF-5 stimulated endochondral ossification can improve scaffold integration in an orthotopic cartilage defect model

This work was aimed at study the role of paraspinal muscles on spinal tensegrity. Four different models of spinal tensegrity breakage with and without injury of the posterior spinal muscle were investigated. Fifteen minipigs (mean age 6-week) underwent costotransversectomy (CTT) at 5 consecutive vertebral segments. In 4 animals ribs and transverse processes (T7–T11) were removed through a posterior midline approach with complete desinsertion of paraspinal muscles. In other 3 animals, CTT was performed by a posterolateral approach (T6–T10) without detachment of paraspinal muscles. Other 4 minipigs underwent rib resection (T7-T11) throughout a thoracoscopic approach avoiding damage of posterior spinal muscles. A final group of 4 animals, a complete detachment of the paraspinal muscles was performed from T7 to T11 without removing bony structures and leaving in deep surgical wax attached to the spinous and transverse processes to avoid reinsertion of the muscles after surgery. Anatomic specimens were radiologically and macroscopically studied just at sacrifice 5 months after surgery. All 4 animals operated on of CTT by midline posterior approach developed structural spinal deformity with curve convexity at the side of rib removal (mean Cobb angle 34,6°). Animals undergoing CTT by posterolateral approach without paraspinal muscle detachment did not develop any significant spinal deformity. Absence of spinal deformity was also found in those animals in which rib resection was performed by thoracoscopy without injury of the posterior spinal muscles. All 4 animals undergoing detachment of the paraspinal muscles without CTT and application of the surgical wax developed scoliotic curves (mean Cobb angle of 28°). In conclusion, a new insight on the underlying pathogenic mechanisms of scoliotic curves is given by using this spinal tensegrity model. Isolated damage of the posterior muscle-ligamentous structures around the costotransverse joints breaking muscles spine tensegrity seems to be mandatory to induce scoliotic deformity. Rib removal alone appeared to have less scoliotic inductive implication. The finding questions previous knowledge on scoliosis etiopathogeny

Anatomisches Institut der Georg-August-Universität Göttingen, Germany. Biomedical Research Centre, Dept. of Orthopaedic Surgery, Academic Hospital, Pretoria, South Africa. To date, no animal model for disc degeneration has gained much acceptance, mostly due to the fact that most animals are quadrupeds and thus lack basic biomechanical characteristics of human spines. An adequate model would be of invaluable interest for degeneration related research. In a standardized series of animal experiments in 18 adult Minipigs and 20 adult Cercopithecus aethiops monkeys all animals obtained nucleotomy in one lumbar FSU from a ret-roperitoneal approach and were sacrificed at last 24 weeks afterwards. The Minipigs were x-rayed at time of sacrifice, the monkeys prior to operation and at termination of the experiment. Vice versa, the Minipigs obtained intradiscal pressure recordings at these occasions. The Minipig spines were formol fixed whereas the monkey spines were harvested after perfusion with PBS, fresh frozen, and obtained CT and MRI scans prior to thawing, fixation and comprehensive histological evaluation. The lumbar FSU of Minipig and Cercopithecus mainly consists of the same elements as in man. There are certain differences concerning the porcine endplates which ossify as an epiphyseal-like formation with ossification starting in its center, different from the so-called “Randleiste”. Whereas the operative procedure in the Minipigs came in handy, in the Cercopithecus monkey it proved to be demanding, though feasible, due to relatively wide transverse processes and thick psoas muscle structures. The psoas could not be easily detached and needed to be split instead, thus directly exposing the segment nerves. The histological, standard radiological, CT, MRI, and mechanical observations were very similar to those which can be made during the natural aging process of the disc in man. Both animal models are recommendable for further research: Cercopithecus FSUs are more difficult to expose. Logistic reasons may favour Minipigs in Europe. In case of fusion related experiments the use of primates yet seems inevitable

Introduction: There is a need for new non-invasive, predictable and quantifiable techniques to assess the process of fracture healing and remodelling in bone. There are several methods to monitor the bone healing in-vivo. But these methods either fail as quantitative predictors of the healing process (X-ray) or exhibit complicated and expensive measurement principles. Some known in-vivo stiffness measurement methods have several disadvantages including the risk of bone malalignment. Therefore we compared ex-vivo torsional strength of bone with in-vivo torsional stiffness under minimal load in two animal model of distraction osteogenesis. Additionally the device was tested in an ex-vivo model. Methods: An external fixator was combined with a rotating double half-ring. The measurement device was fixed to the half-ring during measurements. It was equipped with a linear variable differential transducer, a load cell, and a stepper motor. During measurements the two parts of the half-ring were rotated against each other and the load and displacement were recorded. The slope coefficient after performing a linear regression between data points of moment and displacement curve was defined as stiffness. Afterwards all models were tested in a material testing system as gold standard. This was tested in an in-vivo animal study of tibial distraction (minipigs time of consolidation 10 days/sheeps time of consolidation 50 days). Results: Between in-vivo initial torsional stiffness and torsional strength in minipigs we found a highly significant (p=0.001) coefficient of determination of 0.82, but we found only a poor correlation (p> 0.05) in sheeps. However, the results of the ex-vivo model showed a high precision and accuracy. Discussion: The results of this study suggest that the bone regenerate strength of healing bones can be assessed in-vivo by the presented inital stiffness measurement method in the beginning of an early stage of healing as shown in minipigs. But at the end of the healing period the correlation of strength and stiffness leveled off. There is a similar model showing an excellent correlation, that agree with our data. They explained the weakening of the correlation at the end of healing by a transformation of early bone to lamellar bone after a 2/3 consolidation. In summary, the presented device could be a reliable future tool to monitor the healing progress in patients with bone malalignement or fractures in the beginning of the healing period

Articular cartilage repair remains a challenge in orthopedic surgery, as none of the current clinical therapies can regenerate the functional hyaline cartilage tissue. In this study, we proposed a one-step surgery strategy that uses autologous bone marrow mesenchymal stem cells (MSCs) embedded in type II collagen (Col-II) gels to repair the full thickness chondral defects in minipig models. Briefly, 8 mm full thickness chondral defects were created in both knees separately, one knee received Col-II + MSCs transplantation, while the untreated knee served as control. At 1, 3 and 6 months postoperatively, the animals were sacrificed, regenerated tissue was evaluated by magnetic resonance imaging, macro- and microscopic observation, and histological analysis. Results showed that regenerated tissue in Col-II + MSCs transplantation group exhibited significantly better structure compared with that in control group, in terms of cell distribution, smoothness of surface, adjacent tissue integration, Col-II content, structure of calcified layer and subchondral bone. With the regeneration of hyaline-like cartilage tissue, this one step strategy has the potential to be translated into clinical application

Mesenchymal stem cells (MSC) are promising for the treatment of articular cartilage defects; however, common protocols for in vitro chondrogenesis induce typical features of hypertrophic chondrocytes reminiscent of endochondral bone formation. This may implicate a risk for graft stability. We here analysed the early healing response in experimental full-thickness cartilage defects, asking whether and how MSC can differentiate to chondrocytes in an orthotopic environment. Cartilage defects in knees of minipigs were covered with a collagen-type I/III membrane, and half of them received transplantation of expanded autologous MSC. Integration into surrounding cartilage tissue was poor to moderate after 1 and 3 weeks and no sign of cartilaginous matrix production as indicated by negative safranin-O staining was visible for both groups. At 8 weeks regenerative tissue was integrated into the surrounding tissue and a safranin-O positively stained neocartilage was detectable in 4 tissue regenerates out of 6 in the MSC group compared to 2 out of 6 in the MSC-free group. At 1 and 3 weeks after surgery only marginal Col2A1 and no AGC expression were detectable in both groups. At 8 weeks Col2A1 and AGC levels had significantly increased. Hypertrophic maker induction (Col10A1 and MMP13) was similar in both groups 8 weeks after surgery. Immunostaining for collagen type X, however, was restricted to the regenerative tissue close to the subchondral bone in both groups, while collagen type II staining was detected from below the superficial to the deep zone. Our data provide molecular evidence for spontaneous differentiation of MSC in cartilage and the development of a collagen type II positive, collagen type X negative neocartilage. Whether by remodelling of defect filling tissue collagen type X positive areas will further diminish or even disappear from repair cartilage at later stages has to be evaluated in a longer follow-up study

Purpose of the study: In the growing pig, we have been able to achieve localized control of vertebral body growth by selective destruction ofhte physis using the thermal effect of a laser probe (first part of the study). The purpose of the second part of the study was to evaluate the mechanical effects in terms of 1) intersomatic disc mobility, and 2) bony resistance of the vertebral body and risk of fracture. Material and methods: Thoracotomy was performed on two Yucatan micropigs (group A); a 510 nm 30W diode laser delivered heat applied to nine vertebral bodies. Four months later, the micropigs were sacrificed. Two normal micropigs (group B) served as controls. The specimens were dissected to the intersomatic disc-ligament complex. Three-level vertebral assemblies were thus obtained for mobility tests (flexion-extension, lateral inclination, right-left rotation). Destruction tests were pursued to fracture. Tests were performed with a Zebris 3D motion analyzer. Computed tomography images and histological findings were also assessed. Results: Motion: In group A, when the discal space appeared normal on the specimen, no difference was noted in motion in comparison with group B. Conversely, when imaging demonstrated discal injury, joint stiffness was noted. The destruction tests showed that in group A specimens the fracture did not occur at the zone of lytic bone destruction caused by the heat delivery. Fractures observed were similar in the two groups, including epiphyseal detachements and sagittal fractures of the vertebral bodies. Discussion: Applying laser-delivered heat to the vertebrae in micropigs enabled partial destruction of the physis without injuring the intervertebral disc. Heat delivery induced a modification in vertebral growth. When imaging showed an intact neighboring intervertebral disc, mechanical tests showed normal mobility and resistance. Bone resistance was not diminished. This result is important to consider for the treatment of vertebral osteoid osteomas with laser. Conclusion: Laser application to vertebral bone is a reproducible method which can stop growth of the minipig vertebral bodies without injuring the intervertebral disc and without reducing bone resistance

Summary Statement. A porcine model using Yucatan minipigs was found to be very promising for the investigation of healing around transcutaneous osseointegrated implants. Pigs demonstrated surprising agility and adaptability including the ability to ambulate on three legs during the immediate postoperative period. Introduction. Previous non weight-bearing and weight-bearing caprine, canine and ovine models have evaluated design, material, and biological coating variations in an attempt to improve the wound healing and skin-implant seal around transcutaneous osseointegrated implants. Although these models have primarily been used as a window into the application of transcutaneous osseointegrated implants in humans, some important model characteristics affecting wound healing and infection have been missing including: 1) replication of the physiological tissue response, and 2) availability of a transcutaneous site with sufficient soft tissue coverage. Pig skin, like human, is relatively hairless, tightly attached to the subcutaneous tissue, vascularised by a cutaneous blood supply, and healed by means of epithelialization. Swine have been extensively utilised for superficial and deep wound healing studies and can offer ample soft tissue coverage following a lower limb amputation. Development of a porcine model is important for continued understanding and improvement of weight-bearing transcutaneous osseointegration. Methods. Two male Yucatan mini-pigs (9 months, 36kg) were fit with transcutaneous osseointegrated prostheses using a single-stage transtibial amputation and prosthesis implantation procedure. The endo-prosthesis consisted of a cylindrical intraosseous threaded section and a smooth transcutaneous section. The transcutaneous sections were smooth to promote epithelialization and deter direct skin-implant adhesion. The implants were custom manufactured from medical grade Ti-6Al-4V alloy. The exo-prosthesis, consisting of an adjustable length leg and foot, was attached by clamp to the supercutaneous portion of the implant following either one or two days of sling constraint to limit initial weight-bearing. Various exo-prosthesis designs and configurations were trialed. The animals’ behavior and gait were closely observed. Weight-bearing was monitored using a force plate. At 5 and 8 weeks, clinical, microbiological, and histological data were examined to assess wound healing and infection at the skin-bone-implant interface. Results. The pigs demonstrated surprising agility and adaptability. They were able to successfully ambulate on three legs during the post-op period before weight-bearing was permitted. They adapted quickly to changes in exo-prosthesis design, position, and length. Although bacterial colonization was verified, neither of the animals exhibited clinical signs of infection over the respective eight and five week studies. Histological results indicated that there was no skin to implant adhesion but that epithelial growth was progressing towards the implant in one animal. Healing of the transcutaneous wound site showed substantial progress but a definitive skin seal was non-existent at the eight week time point. Discussion/Conclusion. This is likely the first animal model developed, having soft tissue characteristics similar to those found in humans, in which an axially-loaded, weight-bearing implant was successfully used. Results indicated that this porcine model offers many advantages over previous models for the development, evaluation, and comparison of the various techniques being advocated to achieve successful transcutaneous osseointegration in humans. The Yucatan miniature pig's ability to ambulate on three legs during the immediate post-operative period and quickly adjust to changes in the exo-prosthesis design, coupled with their physiological similarity to humans, makes them a valuable model for future studies

Although osteochondral defects (OCD) following trauma, sport or degenerative diseases occur frequently, healing remains an unresolved clinical problem. These defects seem to appear more often in convex surfaces than in concave ones. In vivo studies have demonstrated the influence of mechanical conditions on osteochondral repair[. 1. ]. However, the influence of the local joint curvature on the mechanical environment as well as the effect of defect fillings on healing remained unknown. We hypothesize that healing of OCD is strongly affected by the local mechanical environment generated after variations in the joint geometry specifically on concave and convex joint surfaces. To study spontaneous repair, OCD (mm, 1.5mm depth) in 18 minipigs were created. Based on this knowledge, a predictive biphasic finite element model for tissue differentiation was created to simulate osteochondral healing. The model was validated by comparison of simulated healing with histological and histomorphometrical outcomes. Differentiation was regulated by the combination of a mechanical stimulus with a factor for differentiation defined for each tissue. The mechanical conditions arising from different predesigned defect fillings have been evaluated: Grafts with 100% (P1) and 50% (P2) of the native subchondral bone stiffness were analyzed. The healing pattern was in general qualitatively comparable to the findings of a gross examination of the histological sections. Generally, the pattern appears to be almost independent of the joint curvature. More hyaline cartilage (HC) was formed in the concave model during simulated healing. The maximum percentage of HC during the simulations was smaller and occurred earlier in the one (27 vs. 40%). In vivo 33% of HC was registered in the 12th week[. 2. ]. Defect filling restoring sub-chondral bone quality (P1) allowed a larger amount of hyaline cartilage formation than a less rigid filling (P2). Until today the more frequent occurrence of OCD at convex joint surfaces reported in the clinical practice has not been related to the local mechanical environment. This study is the first to demonstrate that this may be related to the mechanical stimulus for healing. In fact, during healing simulation HC formation was affected by changes in the joint surface curvature. A continuity of material properties in the layers under an OCD, which operates as basis for the newly formed cartilage, is important for the development of a tissue with adequate mechanical quality for load transmission. Indeed hyaline cartilage formation occurs earlier when P1 as when P2 was used. The use of a predictive tissue differentiation model allows a better understanding of the mechanical aspects of healing. Further analysis is however required before such algorithm may be applied in clinical cases. To consider mechanical factors affecting healing, appear to be of importance