Do stem cells transfected with CXCR4 enhance bone formation in osteoporosis?

Osteoporosis affects bone mass and bone micro-architecture, reducing mechanical strength. SDF-1 and its ligand CXCR4 play significant roles in the migration and engraftment of mesenchymal stem cells (MSCs). The aim of this study was to investigate the effects of CXCR4 transfected MSCs on bone formation in osteopenic rats. The hypothesis was that MSCs genetically modified to over-express CXCR4, would enhance migration of stem cells from osteopenic rats and when injected intravenously in ovariectomised (OVX) rats, would improve bone formation. MSCs were harvested from femora of young, OVX and adult control rats. The differentiation, CXCR4 expression, in vitro migration and phenotypic characteristics of the cells were compared. Although the phenotypic characteristics of cells from all groups of rats were the same, their differentiation capability, CXCR4 expression and migration was significantly different. MSCs were genetically modified to over-express CXCR4 and in vitro migration investigated. It was found that although young MSCs had the highest migration capability (2x more than their uninfected counterparts, p=0.006), the OVX MSCs when transfected with CXCR4 had the most significant migration from their un-transfected counterpart cells(5x more, p=0.025). Additionally, differentiating MSCs to osteoblasts reduced their CXCR4 expression as well as their migration towards SDF1. The CXCR4 transfected MSCs were administered intravenously in OVX rats. Fluorescent labelled cells were tracked after 1 week and were located in the blood vessels of the femur. 11-weeks post-injection, OVX rats injected with young-CXCR4 MSCs had significantly higher BMD(694.0±80.1mg/cm3) (p<0.05) in comparison to rats injected with saline. Rats injected with OVX-CXCR4 MSCs(645.4±79.3mg/ccm) had a higher BMD in comparison to those injected with OVX MSCs(631.4±69.5mg/ccm) and saline(563.4±82.9mg/ccm). The L4 vertebral stiffness was also higher in rats treated with young-CXCR4 MSCs in comparison to those treated with saline. CXCR4 genetically modified MSCs from young and OVX patients may help in boosting bone formation in osteoporosis

Mesenchymal stem cells with increased stromal cell-derived factor 1 expression enhanced fracture healing

Treatment of critical size bone defects pose a challenge in orthopedics. Stem cell therapy together with cytokines has the potential to improve bone repair as they cause the migration and homing of stem cells to the defect site. However, the engraftment, participation, and recruitment of other cells within the regenerating tissue are important. To enhance stem cell involvement, this study investigated overexpression of stem cells with stromal cell-derived factor 1 (SDF-1) using an adenovirus. We hypothesized that these engineered cells would effectively increase the migration of native cells to the site of fracture, enhancing bone repair. Before implantation, we showed that SDF-1 secreted by transfected cells increased the migration of nontransfected cells. In a rat defect bone model, bone marrow mesenchymal stem cells overexpressing SDF-1 showed significantly (p=0.003) more new bone formation within the gap and less bone mineral loss at the area adjacent to the defect site during the early bone healing stage. In conclusion, SDF-1 was shown to play an important role in accelerating fracture repair and contributing to bone repair in rat models, by recruiting more host stem cells to the defect site and encouraging osteogenic differentiation and production of bone

Mesenchymal stromal cells and platelet-rich plasma promote tendon allograft healing in ovine anterior cruciate ligament reconstruction

Purpose The effect of bone marrow mesenchymal stromal cells (BMSCs) and platelet-rich plasma (PRP) on tendon allograft maturation in a large animal anterior cruciate ligament (ACL) reconstruction model was reported for the first time. It was hypothesised that compared with non-augmented ACL reconstruction, BMSCs and PRP would enhance graft maturation after 12 weeks and this would be detected using magnetic resonance imaging (MRI). Methods Fifteen sheep underwent unilateral tendon allograft ACL reconstruction using aperture fixation and were randomised into three groups (n = 5). Group 1 received 10 million allogeneic BMSCs in 2 ml fibrin sealant; Group 2 received 12 ml PRP in a plasma clot injected into the graft and bone tunnels; and Group 3 (control) received no adjunctive treatment. At autopsy at 12 weeks, a graft maturation score was determined by the sum for graft integrity, synovial coverage and vascularisation, graft thickness and apparent tension, and synovial sealing at tunnel apertures. MRI analysis (n = 2 animals per group) of the signal–noise quotient (SNQ) and fibrous interzone (FIZ) was used to evaluate intra-articular graft maturation and tendon–bone healing, respectively. Spearman’s rank correlation coefficient (r) of SNQ, autopsy graft maturation score and bone tunnel diameter were analysed. Results The BMSC group (p = 0.01) and PRP group (p = 0.03) had a significantly higher graft maturation score compared with the control group. The BMSC group scored significantly higher for synovial sealing at tunnel apertures (p = 0.03) compared with the control group. The graft maturation score at autopsy significantly correlated with the SNQ (r = − 0.83, p < 0.01). The tunnel diameter of the femoral tunnel at the aperture (r = 0.883, p = 0.03) and mid-portion (r = 0.941, p = 0.02) positively correlated with the SNQ. Conclusions BMSCs and PRP significantly enhanced graft maturation, which indicates that orthobiologics can accelerate the biologic events in tendon allograft incorporation. Femoral tunnel expansion significantly correlated with inferior maturation of the intra-articular graft. The clinical relevance of this study is that BMSCs and PRP enhance allograft healing in a translational model, and biological modulation of graft healing can be evaluated non-invasively using MRI

CXCR4 has the Potential to Enhance Bone Formation in Osteopenic Rats

Osteoporosis is characterised by reduced bone mass and aberrant bone micro-architecture; thus increasing susceptibility to fracture due to reduced strength and quality. The aims of this study were to investigate the role of CXCR4 transfected on stem cell homing and osteogenic characteristics in osteopenic rats; particularly elucidating the effect on cell migration. METHODS: MSCs were harvested from young, adult and ovarectomized animals and transfected with CXCR4; these cells were administered intravenously in ovarectomized rats. Micro CT and mechanical testing was completed after 12 weeks. RESULTS: rats injected with young CXCR4 transfected cells had significantly higher BMD compared to placebo injected rats (p<0.05). Rats injected with ovarectomized CXCR4 transfected cells, had higher BMD compared to those injected with saline or non-transfected cells (p<0.04). L4 vertebral stiffness was significantly higher in rats treated with young CXCR4 transfected cells compared to all other groups (p<0.05). CONCLUSIONS: CXCR4 genetically modified cells from young and ovarectomized sources improve some aspects of bone formation in the ovarectomized model of osteoporosis; and thus, may play a role in patient treatment regimens

The influence of gap size on the development of fracture union with a micro external fixator

Increasingly, the rat femoral fracture model is being used for preclinical investigations of fracture healing, however, the effect of gap size and its influence on mechanobiology is not well understood. We aimed to evaluate the influence of osteotomy gap on osteotomy healing between the previously published extremes of guaranteed union (0.5 mm) and non-union (3 mm) using this model. A femoral osteotomy in 12–14 week old female Wistar rats was stabilised with a micro fixator (titanium blocks, carbon fiber bars) with an osteotomy gap of 1.0 mm (n = 5), 1.5 mm (n = 7), 2.0 mm (n = 6). After five weeks, the left femur was retrieved. The osteotomy gap was scanned using X-ray microtomography and then histologically evaluated. The radiographic union rate (complete mineralised bone bridging across the osteotomy) was three times higher for the 1.0 mm than the 2.0 mm gap. The 1.0 mm gap had the largest callus (0.069μm3) and bone volume (0.035μm3). Callus and bone volume were approximately 50% smaller within the 2.0 mm gap. Using cadaveric rat femurs stabilised with the external fixator, day 0 mechanical assessment of construct stiffness was calculated on materials testing machine displacement vs load output. The construct stiffness for the 1.0, 1.5 and 2.0 mm gaps was 32.6 ± 5.4, 32.5 ± 2.4, and 32.4 ± 8.3 N/mm (p = 0.779). Interfragmentary strain (IFS) was calculated using the change in osteotomy gap displacement as measured using microstrain miniature differential reluctance transducer spanning the osteotomy gap. Increasing the gap size significantly reduced the IFS (p = 0.013). The mean ‘day 0’ IFS for the 1.0, 1.5 and 2.0 mm gaps were 11.2 ± 1.3, 8.4 ± 1.5 and 6.1 ± 1.2% respectively. A 1.5 mm gap resulted in a delayed fracture healing by 5 weeks and may represent a useful test environment for fracture healing therapy. Increasing gap size did not affect construct stiffness, but did reduce the ‘day 0’ IFS, with a doubling of non-union and halving of bone volume measured between 1.0 and 2.0 mm gaps

Clinical Cohort Study in Canine Patients, to Determine the Average Platelet and White Blood Cell Number and Its Correlation with Patient’s Age, Weight, Breed and Gender: 92 Cases (2019–2020)

Due to its easy preparation and that it is well tolerated, the use of autologous platelet-rich plasma (PRP) has become increasingly popular in regenerative medicine. However, there are still no clear guidelines on how it should be classified or whether the individual canine patient’s clinical status can influence its quality. Objective: This study aims to show if the weight, age, sex, neutered status or breed of canine patients have any correlation with the composition of PRP. Design: A blinded count of the platelets and white blood cells (WBC) was performed from 111 samples from 92 client owned dogs undergoing treatment for degenerative joint disease (DJD). The results were analysed using Pearson correlation test, ANOVA test or Student T-test. Results: There is a positive correlation between the number of platelets and WBC in canine patients of different breeds, but there was no significant difference on the platelet number and WBC number among the different breeds. The weight of the patient is also directly correlated to the platelet number (p = 0.003) but not WBC number. WBC number was negatively correlated to the weight of the patient. The sex and age of the patient did not affect platelets and WBC number, although WBC number is increased in non-neutered male population (p = 0.003). However, it would be interesting to investigate whether the growth factors released from the platelet granules are affected by patient variables in a canine population. Conclusions: Our results show that it is possible to obtain good quality autologous PRP, irrespective of age, sex, neutered status or weight of the patient, for PRP regenerative therapy

Comparison of Fat Harvested from Flank and Falciform Regions for Stem Cell Therapy in Dogs

Background: Adipose tissue has recently gained attention as a source of mesenchymal stem cells (AdMSCs) for applications in treating degenerative joint disease in veterinary patients. This study aimed to quantify the stromal vascular fractions (SVFs) and colony forming units (CFU) of AdMSCs from the falciform and flank regions and compare dogs of different ages and weights. Methods: Fat tissue was harvested from the flank (21 dogs) and falciform regions (17 dogs). The fat tissue was enzymatically digested and the number of nucleated cells in the SVF was counted. The SVF was cultured in vitro and the cell growth was assessed by counting the CFU per gram of fat and the aspect ratio of the cells. Conclusions: There was no significant difference in the number of nucleated cells in the SVF from the two sites. The CFU/g of fat from falciform was 378.9 &plusmn; 293 g and from flank was 486.8 &plusmn; 517 g, and this was also insignificant. Neither age nor weight of the patient had an impact on the SVF or CFU/g. No surgical complications were reported from either of the sites. Harvesting fat for stem cell therapy for intra-articular therapy of degenerative joint disease can be an easy and fast process when obtaining the fat either from the flank or the falciform region, and it is not age or weight dependent. The harvest site for clinical canine patients can be left to the surgeon&rsquo;s discretion and comfort