Are the Properties of Bone Marrow-Derived Mesenchymal Stem Cells Influenced by Overweight and Obesity?

Bone marrow-derived mesenchymal stem cells (BMSCs) are promising candidates for cell-based therapies. Growing evidence has indicated that overweight/obesity can change the bone marrow microenvironment, which affects some properties of BMSCs. As the overweight/obese population rapidly increases, they will inevitably become a potential source of BMSCs for clinical application, especially when receiving autologous BMSC transplantation. Given this situation, the quality control of these cells has become particularly important. Therefore, it is urgent to characterize BMSCs isolated from overweight/obese bone marrow environments. In this review, we summarize the evidence of the effects of overweight/obesity on the biological properties of BMSCs derived from humans and animals, including proliferation, clonogenicity, surface antigen expression, senescence, apoptosis, and trilineage differentiation, as well as the underlying mechanisms. Overall, the conclusions of existing studies are not consistent. Most studies demonstrate that overweight/obesity can influence one or more characteristics of BMSCs, while the involved mechanisms are still unclear. Moreover, insufficient evidence proves that weight loss or other interventions can rescue these qualities to baseline status. Thus, further research should address these issues and prioritize developing methods to improve functions of overweight- or obesity-derived BMSCs.

Osseoperception in transcutaneous osseointegrated prosthetic systems (TOPS) after transfemoral amputation: a prospective study.

BACKGROUND: Endo-exo prosthetics (EEP), which belongs to the transcutaneous osseointegrated prosthetic systems (TOPS), provides an alternative bone-anchored rehabilitation method for transfemoral amputees. It led to the question of whether transmitted forces from prosthetic feet are perceptible by osseoperception resulting in proprioceptive feedback of ground conditions. OBJECTIVES: The following hypotheses emerged for our trial with the null hypothesis: EEP fitting after transfemoral amputation does not influence osseoperception. Alternative hypothesis 1: EEP patients achieve better osseoperception results than transfemoral amputees fitted with socket prosthesis. Alternative hypothesis 2: EEP carriers achieve comparable results with regards to their osseoperception as non-amputees. METHODS: N = 25 patients with EEP (mean age = 50,6 ± 9,4, male/female = 15/10) N = 25 patients with socket prostheses (mean age = 52,6 ± 13,1, male/female = 19/6) and N = 25 healthy volunteers were included in the experimental case-control study. In three blinded test modules (V1, V2, V3), the participants had to identify different degrees of shore hardness (c) of different materials (rubber balls (shore = 5-25c), foam cushions (shore = 5-30c), foam mats (shore = 5-30c) with their prosthetic foot (or a personally defined foot in healthy volunteers) without footwear and had to rank them into the correct order according to their tactile sensation and the degree of hardness. A maximum of 10 points could be scored per run. RESULTS: This experimental observational study included N = 75 participants. The mean age for the entire cohort was 42.8 ± 16.6 years and the BMI was 26.0 ± 4.8. Our results show a significant level of differences in tactile osseoperception between all groups (p < 0.001). A correlation between the mean values of V1-3 and the PMQ2.0 as well as the mean values of K-Level and the prosthesis wearing time per day showed for PMQ (r = 0.387, p = 0.006) and K-level (r = 0.448, p = 0.001) which is a moderate effect according to Cohen. CONCLUSION: Our study results suggest that the EEP treatment can lead to an improvement in tactile sensory perception via the bone-anchored implant, which can lead to an increase in quality of life and improved gait safety.

Influence of age on stem cells depends on the sex of the bone marrow donor.

Ageing is often accompanied by an increase in bone marrow fat together with reduced bone volume and diseases of the bone such as osteoporosis. As mesenchymal stem cells (MSCs) are capable of forming bone, cartilage and fat tissue, studying these cells is of great importance to understand the underlying mechanisms behind age-related bone diseases. However, inter-donor variation has been found when handling MSCs. Therefore, the aim of this study was to investigate the effects of donor age and sex by comparing in vitro characteristics of human bone marrow-derived MSCs (hBMSCs) from a large donor cohort (n = 175). For this, hBMSCs were analysed for CFU-F capacity, proliferation, differentiation capacity and surface antigen expression under standardized culture conditions. The results demonstrated a significantly reduced CFU-F number for hBMSCs of female compared to male donors. Furthermore, there was a significant decrease in the proliferation rate, adipogenic differentiation potential and cell surface expression of SSEA-4, CD146 and CD274 of hBMSCs with an increase in donor age. Interestingly, all these findings were exclusive to hBMSCs from female donors. Further research should focus on postmenopausal-related effects on hBMSCs, as the results imply a functional loss and immunophenotypic change of hBMSCs particularly in aged women.

Altered bone development with impaired cartilage formation precedes neuromuscular symptoms in spinal muscular atrophy.

Spinal muscular atrophy (SMA) is a fatal neurodegenerative disease of newborns and children caused by mutations or deletions of the survival of motoneuron gene 1 resulting in low levels of the SMN protein. While neuromuscular degeneration is the cardinal symptom of the disease, the reduction of the ubiquitously expressed SMN additionally elicits non-motoneuron symptoms. Impaired bone development is a key feature of SMA, but it is yet unknown whether this is an indirect functional consequence of muscle weakness or caused by bone-intrinsic mechanisms. Therefore, we radiologically examined SMA patients in a prospective, non-randomized cohort study characterizing bone size and bone mineral density (BMD) and performed equivalent measurements in pre-symptomatic SMA mice. BMD as well as lumbar vertebral body size were significantly reduced in SMA patients. This growth defect but not BMD reduction was confirmed in SMA mice by µCT before the onset of neuromuscular symptoms indicating that it is at least partially independent of neuromuscular degeneration. Interestingly, the number of chondroblasts in the hypertrophic zone of the growth plate was significantly reduced. This was underlined by RNAseq and expression data from developing SMA mice vertebral bodies, which revealed molecular changes related to cell division and cartilage remodeling. Together, these findings suggest a bone intrinsic defect in SMA. This phenotype may not be rescued by novel drugs that enhance SMN levels in the central nervous system only.

Suppression of the interleukin-1ß-induced inflammatory response of human Chang liver cells by acute and subacute exposure to alcohol: an in vitro study.

AIM: To evaluate protective immunosuppressive dose and time-dependent effects of ethanol in an in vitro model of acute inflammation in human Chang liver cells. METHOD: The study was performed in 2016 and 2017 in the research laboratory of the Department of Trauma, Hand and Reconstructive Surgery, the University Hospital of the Goethe-University Frankfurt. Chang liver cells were stimulated with either interleukin (IL)-1ß or IL-6 and subsequently treated with low-dose ethanol (85 mmol/L) or high-dose ethanol (170 mmol/L) for one hour (acute exposure) or 72 hours (subacute exposure). IL-6 and IL-1ß release were determined by enzyme-linked immunosorbent assay. Neutrophil adhesion to Chang liver monolayers, production of reactive oxygen species, and apoptosis or necrosis were analyzed. RESULTS: Contrary to high-dose ethanol, acute low-dose ethanol exposure significantly reduced IL-1ß-induced IL-6 and IL-6-induced IL-1ß release (P<0.05). Subacute ethanol exposure did not change proinflammatory cytokine release. Acute low-dose ethanol exposure significantly decreased inflammation-induced formation of reactive oxygen species (P<0.05) and significantly improved cell survival (P<0.05). Neither acute nor subacute high-dose ethanol exposure significantly changed inflammation-induced changes in reactive oxygen species or survival. Acute and subacute ethanol exposure, independently of the dose, significantly decreased neutrophil adhesion to inflamed Chang liver cells (P<0.05). CONCLUSION: Acute treatment of inflamed Chang liver cells with ethanol showed its immunosuppressive potential. However, the observed effects were limited to low-dose setting, indicating the relevance of ethanol dose in the modulation of inflammatory cell response.

A comparative study of bone biopsies from the iliac crest, the tibial bone, and the lumbar spine.

BACKGROUND: Patients with an impaired renal function show a high incidence of bone and mineral disturbances. These 'chronic kidney disease - mineral and bone disorders' (CKD-MBD) range from high turnover osteoporosis to adynamic bone disease. Currently, the histomorphometric analysis of a bone biopsy taken from the iliac crest is viewed as the gold standard for CKD-MBD subtype differentiation. However, the clinical relevance of such a biopsy is questionable since iliac crest fractures are an extremely rare finding. Therefore, we aimed to elucidate if the histomorphometric parameter 'trabecular bone volume (BV/TV)' from the iliac crest is representative for other biopsy locations. We chose two skeletal sites of higher fracture risk for testing, namely, the tibial bone and the lumbar spine, to examine if the current gold standard of bone biopsy is indeed golden. METHODS: Bone biopsies were taken from 12 embalmed body donors at the iliac crest, the proximal tibia, and the lumbar vertebral body, respectively. Masson-Goldner stained sections of methyl methacrylate embedded biopsies were used for trabecular bone volume calculation. Furthermore, exemplary µ-computed tomography (XtremeCT) scans with subsequent analysis were performed. RESULTS: Median values of trabecular bone volume were comparable between all body donors with median (interquartile range, IQR) 18.3% (10.9-22.9%) at the iliac crest, 21.5% (9.5-40.1%) at the proximal tibia, and 16.3% (11.4-25.0%) at the lumbar spine. However, single values showed extensive intra-individual variation, which were also confirmed by XtremeCT imaging. CONCLUSIONS: Distinct intra-individual heterogeneity of trabecular bone volume elucidate why a bone biopsy from one site does not necessarily predict patient relevant endpoints like hip or spine fractures. Physicians interpreting bone biopsy results should know this limitation of the current gold standard for CKD-MBD diagnostic, especially, when systemic therapeutic decisions should be based on it.

Severe Hemorrhagic Shock Leads to a Delayed Fracture Healing and Decreased Bone Callus Strength in a Mouse Model.

BACKGROUND: Multiple trauma is frequently associated with hemorrhagic shock and fractures of the extremities. Clinically, the rate of impaired fracture healing (delayed healing and nonunion) seems to be increased in patients with multiple injuries compared with patients with isolated fractures. As the underlying pathogenesis remains poorly understood, we aimed to analyze the biomechanical properties during fracture healing in a murine model. QUESTIONS: The aim of this study was to determine whether fracture healing after severe hemorrhagic shock results in (1) delayed bridging as determined by macroscopic and radiographic assessment, (2) altered conditions of callus components as determined by µCT, and (3) decreased maximum bending moment measured by a three-point-bending test compared with ordinary fracture healing. METHODS: Male C57BL/6NCrl mice were randomly assigned to five groups and four different times (five to 10 mice per group and time). Only the right femur from each mouse was used for analysis: the trauma hemorrhage (TH) group received a pressure-controlled hemorrhagic shock via catheter; the osteotomy (Fx) group underwent osteotomy and implantation of an external fixator on the right femur; the combined trauma (THFx) group received hemorrhagic shock and an external fixator with osteotomy; the sham group underwent implantation of a catheter and external fixator but had no blood loss or osteotomy, and the control group underwent no interventions. After 2, 3, 4, or 6 weeks, five to 10 animals of each group were sacrificed. Bones were analyzed macroscopically and via radiographs, µCT, and three-point-bending test. Statistical significance was set at a probability less than 0.05. Comparisons were performed using the Mann-Whitney U or the Kruskal-Wallis test. RESULTS: In the Fx group, the osteotomy gap was stable and bridged after 2 weeks in contrast to some bones in the THFx group where stable bridging did not occur. No difference was observed between the groups. µCT analysis showed reduced density of bone including callus (THFx: 1.17 g/cm3; interquartile range [IQR], 0.04 g/cm3; Fx: 1.22 g/cm3; IQR, 0.04 g/cm3; p = 0.002; difference of medians [DM], -0.048; 95% CI, -0.073 to -0.029) and increased share of callus per volume of bone mass (%) after 2 weeks in the THFx group compared with the Fx group (THFx: 44.16%; IQR, 8.66%; Fx: 36.73%; IQR, 4.39%; p = 0.015; DM, 7.634; 95% CI, 2.018-10.577). The three-point-bending test established a decreased maximum bending moment in the THFx group compared with the Fx group 2 weeks after surgery (THFx: 7.10 Nmm; IQR, 11.25 Nmm; Fx: 11.25 Nmm; IQR, 5.70 Nmm; p = 0.026; DM, -5.043; 95% CI, -10.867 to -0.74). No differences were observed between the THFx and Fx groups after more than 2 weeks. CONCLUSION: In this in vivo mouse fracture model, we conclude that hemorrhagic shock retards fracture healing during the early phase of the facture healing process. CLINICAL RELEVANCE: A severe hemorrhagic shock in patients could result in initial delayed fracture healing and needs special attention. We plan to conduct a prospective, observational clinical research study to analyze if delayed fracture healing occurs in patients after severe blood loss.

A new multiple trauma model of the mouse.

BACKGROUND: Blunt trauma is the most frequent mechanism of injury in multiple trauma, commonly resulting from road traffic collisions or falls. Two of the most frequent injuries in patients with multiple trauma are chest trauma and extremity fracture. Several trauma mouse models combine chest trauma and head injury, but no trauma mouse model to date includes the combination of long bone fractures and chest trauma. Outcome is essentially determined by the combination of these injuries. In this study, we attempted to establish a reproducible novel multiple trauma model in mice that combines blunt trauma, major injuries and simple practicability. METHODS: Ninety-six male C57BL/6 N mice (n = 8/group) were subjected to trauma for isolated femur fracture and a combination of femur fracture and chest injury. Serum samples of mice were obtained by heart puncture at defined time points of 0 h (hour), 6 h, 12 h, 24 h, 3 d (days), and 7 d. RESULTS: A tendency toward reduced weight and temperature was observed at 24 h after chest trauma and femur fracture. Blood analyses revealed a decrease in hemoglobin during the first 24 h after trauma. Some animals were killed by heart puncture immediately after chest contusion; these animals showed the most severe lung contusion and hemorrhage. The extent of structural lung injury varied in different mice but was evident in all animals. Representative H&E-stained (Haematoxylin and Eosin-stained) paraffin lung sections of mice with multiple trauma revealed hemorrhage and an inflammatory immune response. Plasma samples of mice with chest trauma and femur fracture showed an up-regulation of IL-1ß (Interleukin-1ß), IL-6, IL-10, IL-12p70 and TNF-α (Tumor necrosis factor- α) compared with the control group. Mice with femur fracture and chest trauma showed a significant up-regulation of IL-6 compared to group with isolated femur fracture. CONCLUSIONS: The multiple trauma mouse model comprising chest trauma and femur fracture enables many analogies to clinical cases of multiple trauma in humans and demonstrates associated characteristic clinical and pathophysiological changes. This model is easy to perform, is economical and can be used for further research examining specific immunological questions.

[Non-cryoconserving storage strategies for fresh osteochondral allografts]. / Nichtkryokonservierende Lagerungsstrategien für frische osteochondrale Allografts.

BACKGROUND: The clinical outcome of fresh allogeneic osteochondral allografts (OCA) is greatly dependent on the number of viable chondrocytes at the time of implantation. The selection and preparation of a suitable recipient can be very time-consuming and the number of tissue donors is greatly limited; therefore, the preservation of high allograft viability before transplantation is a focal point of current research. OBJECTIVE: The objective of this review is to give an overview of established storage strategies for OCA and to serve as a decision-making aid for German clinics in the choice of a suitable storage strategy. MATERIAL AND METHODS: A search of the literature published between January 2002 and May 2017 was independently performed by two persons with respect to original works on storage strategies of OCA with a focus on storage medium, use of fetal bovine serum, storage temperature and change of medium. A total of 20 suitable studies were selected for this review. RESULTS: Based on the current studies a clearly superior storage solution could not be identified; however, storage at 4 °C seems to give better results with respect to cell viability than storage at 37 °C. High chondrocyte viability rates after 28 days of storage were also achieved using media without the addition of fetal bovine serum. CONCLUSION: A major difficulty in comparing the relevant studies on storage solutions is that multiple aspects in the study design varied between the studies. Due to this no definite conclusion on what the ideal storage strategy should look like could be drawn. Further studies are needed to conclusively show whether cell culture medium-based storage solutions are truly superior to those based on Ringer-lactate solutions.

Identification of potential biomarkers for post-traumatic complications released after trauma-hemorrhage from murine Kupffer cells and its investigation in lung and liver.

CONTEXT: Early diagnosis of complications after severe trauma by specific biomarkers remains difficult. OBJECTIVE: Identify potential new biomarkers for early diagnosis of post-traumatic complications. MATERIAL AND METHODS: Mice underwent pressure-controlled hemorrhage or sham procedure. Four hours later, genome-wide expression of isolated Kupffer cells was compared with controls using Affymetrix-Genechip-Expression-Analysis and real-time-PCR. RESULTS: Expression analysis and real-time-PCR revealed a significant increase of gene expression of Cxcl10, Il4ra, Csf2rb2, Lcn2, and Gbp5. CONCLUSION: Cxcl10, Il4ra, Csf2rb2, Lcn2, and Gbp5 might represent new biomarkers for early diagnosis of post-traumatic complications, if they are linked to the development of post-traumatic complications.

Chondrocyte survival in osteochondral transplant cylinders depends on the harvesting technique.

PURPOSE: In autologous osteochondral transplantation, the edges of the harvested plug are particularly susceptible to mechanical or thermal damage to the chondrocytes. We hypothesised that the applied harvesting device has an impact on chondrocyte vitality. METHODS: Both knees of five blackhead sheep (ten knees) underwent open osteochondral plug harvesting with three different circular harvesting devices (osteoarticular transfer system harvester [OATS; diameter 8 mm; Arthrex, Munich, Germany], diamond cutter [DC; diameter 8.35 mm; Karl Storz, Tuttlingen, Germany] and hollow reamer with cutting crown [HRCC; diameter 7 mm; Dannoritzer, Tuttlingen, Germany]) from distinctly assigned anatomical sites of the knee joint. The rotary cutters (DC and HRCC) were either used with (+) or without cooling (-). Surgical cuts of the cartilage with a scalpel blade were chosen as control method. After cryotomy cutting, chondrocyte vitality was assessed using fluorescence microscopy and a Live/Dead assay. RESULTS: There were distinct patterns of chondrocyte vitality, with reproducible accumulations of dead chondrocytes along the harvesting edge. No statistical difference in chondrocyte survivorship was seen between the OATS technique and the control method, or between the HRCC+ technique and the control method (P > 0.05). The DC+, HRCC- and DC- techniques yielded significantly lower chondrocyte survival rates compared with the control method (P < 0.05). CONCLUSIONS: Chondrocyte survival in osteochondral cylinders depends on the applied harvesting technique. The use of rotary cutters without cooling yielded worst results, while the traditional OATS punch and rotary cutters with cooling achieved comparable rates of chondrocyte vitality.

Beneficial effects of finasteride on hepatic and pulmonary immune response after trauma hemorrhage in mice.

UNLABELLED: The literature reveals evidence for a gender specific outcome after major trauma and hemorrhage. Increased levels of male sex hormones such as testosterone and even more dihydrotestosterone (DHT) mediate negative effects on the posttraumatic immune response. Pretreatment with finasteride several days before trauma hemorrhage (TH) led to improved outcomes in mice. We hypothesized that finasteride mediates its protective effects also when administered after TH within the resuscitation process. METHODS: Male C57BL/6N-mice underwent TH (blood pressure: 35 mmHg, 90 min) followed by finasteride application and fluid resuscitation. Plasma cytokines (MIP-1ß, TNF-α, MCP-1, MCP-3, IL-6), productive capacity of alveolar macrophages (AM) and hepatic Kupffer cells (KC) and systemic DHT levels were determined 4 h and 24 h thereafter. Pulmonary and hepatic infiltration of PMN was determined by immunohistochemical staining. RESULTS: Finasteride treatment resulted in reduced levels of systemic cytokines. This was accompanied by a reduced posttraumatic cytokine secretion of AM as well as Kupffer cells, thereby reducing hepatic distant organ damage as measured by reduced PMN infiltration. Systemic DHT levels were decreased following finasteride treatment. CONCLUSION: Finasteride exerts salutary effects on the pulmonary and hepatic immune response using a therapeutic approach following TH in mice. Therefore, finasteride might represent a potential agent following multiple trauma and hemorrhage.

The antimicrobial peptide lysozyme is induced after multiple trauma.

The antimicrobial peptide lysozyme is an important factor of innate immunity and exerts high potential of antibacterial activity. In the present study we evaluated the lysozyme expression in serum of multiple injured patients and subsequently analyzed their possible sources and signaling pathways. Expression of lysozyme was examined in blood samples of multiple trauma patients from the day of trauma until 14 days after trauma by ELISA. To investigate major sources of lysozyme, its expression and regulation in serum samples, different blood cells, and tissue samples were analysed by ELISA and real-time PCR. Neutrophils and hepatocytes were stimulated with cytokines and supernatant of Staphylococcus aureus. The present study demonstrates the induction and release of lysozyme in serum of multiple injured patients. The highest lysozyme expression of all tested cells and tissues was detected in neutrophils. Stimulation with trauma-related factors such as interleukin-6 and S. aureus induced lysozyme expression. Liver tissue samples of patients without trauma show little lysozyme expression compared to neutrophils. After stimulation with bacterial fragments, lysozyme expression of hepatocytes is upregulated significantly. Toll-like receptor 2, a classic receptor of Gram-positive bacterial protein, was detected as a possible target for lysozyme induction.

Transcutaneous electromagnetic induction heating of an intramedullary nickel-titanium shape memory implant.

PURPOSE: Inadequate mechanical stimuli are a major cause for nonunions following surgery for femoral and tibial shaft fractures. Adapting fixation rigidity during the course of fracture healing requires additional surgery. Nickel-titanium (NiTi) implants can change shape and rigidity by employing a temperature-dependent shape-memory effect. As a first step in the development of advanced intramedullary (IM) NiTi devices for fracture healing, this study aimed to test the feasibility and safety of transcutaneous electromagnetic induction heating of an IM NiTi implant in a rat model. METHODS: In 51 rats, NiTi implants were introduced into the left distal femur. Forty-four animals were transferred to an induction coil, and the implant was electromagnetically heated to temperatures between 40° and 60 °C Blood samples were drawn before and four hours after the procedure. Interleukin (IL)-1, IL-4, IL-10, tumour necrosis factor alpha (TNF-α) and interferon gamma (IFN-γ) were measured. Animals were sacrificed at three weeks. Histological specimens from the hind leg and liver were retrieved and examined for inflammatory changes, necrosis or corrosion pits. RESULTS: All animals successfully underwent the surgical procedure. Following transcutaneous induction heating, target temperature was confirmed in 37/44 rats. Postoperative controls showed no signs of undue limitations. Neither cytokine measurements nor histological specimens showed any significant differences between groups. There were no corrosion pits or necrosis. CONCLUSION: We conclude that electromagnetic induction heating of IM NiTi implants is feasible and safe in a rat femur model. These findings reflect a further step in the development of novel concepts for IM fracture fixation that might lead to better fracture healing, less patient discomfort and less need for surgical interventions.

Effect of High BMI on Human Bone Marrow-Derived Mesenchymal Stromal Cells.

Bone marrow-derived mesenchymal stromal cells (BMSCs) are attractive candidates in tissue engineering and regenerative medicine. Growing evidence has suggested that a high body mass index (BMI) can affect the properties of BMSCs, resulting in a reduced quality of the cells. However, the results are not consistent. Therefore, this study aimed to investigate the influences of high BMI on human BMSCs (hBMSCs). To avoid gender bias, BMSCs from females and males were studied independently. Finally, hBMSCs from 89 females and 152 males were separately divided into the normal BMI group (18.5 kg/m2 ≤ BMI < 25 kg/m2) and the high BMI group (BMI > 25 kg/m2). The cells were analyzed for the colony-forming potential; proliferation capacity; in vitro adipogenic, osteogenic, and chondrogenic differentiation potentials; and the expression of 32 common surface antigens. The results showed that high BMI did not change the number of colonies at passage 1 in females and males. In contrast, significantly reduced colony numbers at passage 4 (P4) were found in both female and male donors with high BMI. The doubling time of hBMSCs was comparable between the normal and the high BMI groups of females and males. Furthermore, the results of trilineage differentiation did not differ between the different BMI groups of males. In females, the high and the normal BMI groups also showed similar adipogenic and chondrogenic differentiation, while osteogenic differentiation was significantly enhanced in the high-BMI group. Regarding the expression of surface antigens, the expressions of CD200 and SSEA4 on hBMSCs were reduced in the high-BMI group of females and males, respectively. In conclusion, high BMI suppressed the clonogenicity of female and male hBMSCs at P4, improved the in vitro osteogenesis of female hBMSCs, and decreased the expressions of CD200 on hBMSCs in females and SSEA4 in males.

Ex vivo storage of human osteochondral allografts: Long-term analysis over 300 days using a Ringer-based solution.

Large osteochondral defects are a major challenge in orthopedics, for which osteochondral allograft (OCA) transplantation is nowadays considered as an option, especially in young patients. However, a major issue with OCA is the need for graft storage, which ensures adequate cartilage integrity over time. The aim of this study was to test how long a Ringer-based storage solution can provide good graft quality after explantation and thus meet the requirements for OCA. For this purpose, human osteochondral allografts of the knee and ankle were analyzed. Live/Dead analysis was performed and glycosaminoglycan, as well as hydroxyproline content, were measured as crucial chondrocyte integrity factors. Furthermore, biomechanical tests focusing on stress relaxation and elastic compression modulus were performed. The critical value of 70% living chondrocytes, which corresponds to a number of 300 cells/mm², was reached after an average of 16 weeks of storage. In addition, a constant cell shrinkage was observed over time. The amount of glycosaminoglycan and hydroxyroline showed a slight and constant decrease over time, but no significant differences when compared from Day 0 to the values at Weeks 40-43. Biomechanical testing also revealed no significant differences at the different time points. Therefore, the results show that the Ringer-based storage solution at 4°C is able to provide a chondrocyte survival of 70% until Week 16. This is comparable to previously published storage solutions. Therefore, the study contributes to the establishment of a Ringer-based osteochondral allograft transplantation system for countries where medium-based storage solution cannot be approved.

Fracture Healing in Elderly Mice and the Effect of an Additional Severe Blood Loss: A Radiographic and Biomechanical Murine Study.

Femoral fractures and severe bleeding frequently occur in old patients showing a delayed healing. As there are no studies investigating the combined effect of high age and severe blood loss on fracture healing, this was examined radiographically and biomechanically in this study. Therefore, young and old male mice were randomly assigned to three operation groups. In the fracture group (Fx), external fixator and osteotomy were applied to the femur. The combined trauma group (THFx) additionally received a pressure-controlled hemorrhage. Sham animals were only implanted with arterial catheter and external fixator. Sacrifice was performed after three weeks and bone healing was evaluated radiologically via µCT, as well as biomechanically using a three-point bending test. A decreased share of callus/total bone volume was observed in old mice with blood loss compared to old Fx. Hemorrhagic shock also reduced the trabecular number in old mice compared to Fx and young THFx. Moreover, a lower elastic limit in old Sham mice without fracture was revealed. Fracture combined with a high loss of blood further reduced the elastic limit in old mice compared to isolated Fx in old animals. In conclusion, this study showed that severe blood loss has a higher negative effect in old mice compared to young ones.

Characterization of Human, Ovine and Porcine Mesenchymal Stem Cells from Bone Marrow: Critical In Vitro Comparison with Regard to Humans.

For research and clinical use of stem cells, a suitable animal model is necessary. Hence, the aim of this study was to compare human-bone-marrow-derived mesenchymal stem cells (hBMSCs) with those from sheep (oBMSCs) and pigs (pBMSCs). The cells from these three species were examined for their self-renewal potential; proliferation potential; adhesion and migration capacity; adipogenic, osteogenic and chondrogenic differentiation potential; and cell morphology. There was no significant difference between hBMSCs and pBMSCs in terms of self-renewal potential or growth potential. The oBMSCs exhibited a significantly higher doubling time than hBMSCs from passage 7. The migration assay showed significant differences between hBMSCs and pBMSCs and oBMSCs-up to 30 min, hBMSCs were faster than both types and after 60 min faster than pBMSCs. In the adhesion assay, hBMSCs were significantly better than oBMSCs and pBMSCs. When differentiating in the direction of osteogenesis, oBMSCs and pBMSCs have shown a clearer osteogenic potential. In all three species, adipogenesis could only be evaluated qualitatively. The chondrogenic differentiation was successful in hBMSCs and pBMSCs in contrast to oBMSCs. It is also important to note that the cell size of pBMSCs was significantly smaller compared to hBMSCs. Finally, it can be concluded that further comparative studies are needed to draw a clear comparison between hBMSCs and pBMSCs/oBMSCs.