Does size difference in allogeneic cancellous bone granules loaded with differentiated autologous cultured osteoblasts affect osteogenic potential?

We study the efficacy of bone regeneration by using two differently sized allogeneic cancellous bone granules loaded with autologous cultured osteoblasts in a rabbit model. Critical-sized bone defects of the radial shaft were made in 40 New Zealand White rabbits. Small allogeneic bone granules (150–300 μm in diameter) loaded with cultured differentiated autologous osteoblasts were implanted into one forearm (SBG group) and large bone granules (500–710 μm) loaded with osteoblasts were implanted into the forearm of the other side (LBG group). Radiographic evaluations were performed at 3, 6, 9 and 12 weeks and histology and micro-CT image analysis were carried out at 6 and 12 weeks post-implantation. On radiographic evaluation, the LBG group showed a higher bone quantity index at 3 and 6 weeks post-implantation (P?<?0.05) but statistical significance was lost at 9 and 12 weeks. The progression of biological processes of the SBG group was faster than that of the LBG group. On micro-CT image analysis, the LBG group revealed a higher total bone volume and surface area than the SBG group at 6 weeks (P?<?0.05) but the difference decreased at 12 weeks and was without statistical significance. Histological evaluation also revealed faster progression of new bone formation and maturation in the SBG group. Thus, the two differently sized allogeneic bone granules loaded with co-cultured autologous osteoblasts show no differences in the amount of bone regeneration, although the SBG group exhibits faster progression of bone regeneration and remodeling. This method might therefore provide benefits, such as a short healing time and easy application in an injectable form, in a clinical setting.     

Bone regeneration potential of allogeneic or autogeneic mesenchymal stem cells loaded onto cancellous bone granules in a rabbit radial defect model

For developing a clinically effective bone regeneration strategy, we compare the bone regeneration potential of cultured allogeneic bone marrow-derived mesenchymal stem cells (BM-MSCs) and of autologous BM-MSCs loaded onto allogeneic cancellous bone granule scaffolds. A critical-sized segmental bone defect was made at the mid-shaft of both radiuses in 19 New Zealand White rabbits (NWRs). In the experimental group, allogeneic BM-MSCs loaded onto small-sized allogeneic cancellous bone granules (300~700 um in diameter) were implanted in one side of a bone defect. In the control group, autologous BM-MSCs loaded onto allogeneic cancellous granules were grafted in the other side. Bone regeneration was assessed by radiographic evaluation at 4, 8, 12 and 16 weeks post-implantation and by micro-computed tomography (micro-CT) and histological evaluation at 8 and 16 weeks. The experimental groups showed lower bone quantity indices (BQIs) than the control groups at 12 and 16 weeks (p?<?0.05), although no significant difference was observed at 4 and 8 weeks (p?>?0.05). Micro-CT analysis revealed that both groups had similar mean total bone volume and other parameters including trabecular thickness, number and separation at either 8 or 16 weeks. Only bone surface area revealed less area in the experimental group at 16 weeks. Histological evaluation of 8-week and 16-week specimens showed similar biologic processes of new bone formation and maturation. There was no inflammatory reaction indicating an adverse immune response in both allogeneic and autologous MSC groups. In conclusion, allogeneic BM-MSCs loaded onto allogeneic cancellous bone granules had comparable bone regeneration potential to autologous BM-MSCs in a rabbit radial defect model.     

Treatment of long tubular bone defect of rabbit using autologous cultured osteoblasts mixed with fibrin

The osteogenic potential of autologous cultured osteoblasts mixed with fibrin when transplanted to bone defects was evaluated. Radial shaft defects over 15 mm were made in 30 New Zealand white rabbits. A total of 15 rabbits in the control group underwent an iliac bone graft and 15 rabbits in the experimental group underwent an autologous cultured osteoblast injection mixed with fibrin. Both groups were compared radiologically and 5 rabbits in each group were sacrificed for histological evaluation using H-E and Masson’s trichrome stain at 3, 6, and 9 weeks. Osteogenesis in the control group progressed more rapidly than in the experimental group. However, at 9 weeks, bone formation in both groups were similar and showed no significant difference in terms of the amount of bone formation and the quality of bone union. Autologous cultured osteoblast transplantation mixed with fibrin in bone defects was found to produce bone efficiently.     

Reconstruction of Beagle Hemi-Mandibular Defects with Allogenic Mandibular Scaffolds and Autologous Mesenchymal Stem Cells

Objective

Massive bone allografts are frequently used in orthopedic reconstructive surgery, but carry a high failure rate of approximately 25%. We tested whether treatment of graft with mesenchymal stem cells (MSCs) can increase the integration of massive allografts (hemi-mandible) in a large animal model.

Methods

Thirty beagle dogs received surgical left-sided hemi-mandibular defects, and then divided into two equal groups. Bony defects of the control group were reconstructed using allografts only. Those of the experimental group were reconstructed using allogenic mandibular scaffold-loaded autologous MSCs. Beagles from each group were killed at4 (n = 4), 12 (n = 4), 24 (n = 4) or 48 weeks (n = 3) postoperatively. CT and micro-CT scans, histological analyses and the bone mineral density (BMD) of transplants were used to evaluate defect reconstruction outcomes.

Results

Gross and CT examinations showed that the autologous bone grafts had healed in both groups. At 48 weeks, the allogenic mandibular scaffolds of the experimental group had been completely replaced by new bone, which has a smaller surface area to that of the original allogenic scaffold, whereas the scaffold in control dogs remained the same size as the original allogenic scaffold throughout. At 12 weeks, the BMD of the experimental group was significantly higher than the control group (p<0.05), and all micro-architectural parameters were significantly different between groups (p<0.05). Histological analyses showed almost all transplanted allogeneic bone was replaced by new bone, principally fibrous ossification, in the experimental group, which differed from the control group where little new bone formed.

Conclusions

Our study demonstrated the feasibility of MSC-loaded allogenic mandibular scaffolds for the reconstruction of hemi-mandibular defects. Further studies are needed to test whether these results can be surpassed by the use of allogenic mandibular scaffolds loaded with a combination of MSCs and osteoinductive growth factors.     

Local administration of allogeneic or autologous bone marrow-derived mesenchymal stromal cells enhances bone formation similarly in distraction osteogenesis

Background aimsDistraction osteogenesis (DO) is a surgical technique to promote bone regeneration that requires a long time for bone healing. Bone marrow-derived mesenchymal stromal cells (MSCs) have been applied to accelerate bone formation in DO. Allogeneic MSCs are attractive, as they could be ready to use in clinics. Whether allogeneic MSCs would have an effect similar to autologous MSCs with regard to promoting bone formation in DO is still unknown. This study compares the effect of autologous MSCs versus allogeneic MSCs on bone formation in a rat DO model.MethodsRat bone marrow-derived MSCs were isolated, characterized and expanded in vitro. Adult rats were subjected to right tibia transverse osteotomy. On the third day of distraction, each rat received one injection of phosphate-buffered saline (PBS), autologous MSCs or allogeneic MSCs at the distraction site. Tibiae were harvested after 28 days of consolidation for micro-computed tomography examination, mechanical test and histological analysis.ResultsResults showed that treatment with both allogeneic and autologous MSCs promoted bone formation, with significantly higher bone mass, mechanical properties and mineral apposition rate as well as expression of angiogenic and bone formation markers at the regeneration sites compared with the PBS-treated group. No statistical difference in bone formation was found between the allogeneic and autologous MSC treatment groups.ConclusionsThis study indicates that allogeneic and autologous MSCs have a similar effect on promoting bone consolidation in DO. MSCs from an allogeneic source could be used off-the-shelf with DO to achieve early bone healing.     

Mineral status and mechanical properties of cancellous bone exposed to hydrogen peroxide for various time periods

Processed cancellous bone has been regarded as one alternative for the treatment of bone defects. In order to avoid immunogenic effects and preserve the natural properties of the bone, the optimal processing method should be determined. To observe the influence of hydrogen peroxide on the mineral status and mechanical properties of cancellous bone for various time periods and find the optimal processing time. Cancellous bone granules from bovine femur condyles were treated with 30% hydrogen dioxide for 0, 12, 24, 36, 48, 60 and 72 h separately. The microstructure and mineral content of the granules were evaluated by ash analysis, Micro-CT, scanning electron micrograph and energy dispersive X-ray. The biomechanical properties were analyzed by applying cranial-caudal compression in a materials testing machine. With increasing exposure to hydrogen peroxide, the BMD and BMC of granules gradually decreased, and the Ca/P molar ratios clearly increased (P < 0.05). Meanwhile, the mineral content of the granules increased from 48.5 ± 1.3 to 79.5 ± 2.1%. Substantial decreases in the strength of the granules were observed, and after 48 h severe decreases were noted. The decrease in strength was also evident after normalizing the parameters to the cross-sectional area. Granules of bovine cancellous bone matrix should be processed by hydrogen peroxide for 12 to 36 h to fulfill the basic requirements of a bone tissue engineering scaffold. These granules could potentially be useful during orthopedic operations.     

Hemoglobin Regeneration Efficiency in Anemic Rats: Effects on Bone Mineral Composition and Biomechanical Properties

This study reports the effects of dietary iron (Fe) deficiency and recovery on bone mineral composition and strength in anemic rats submitted to a hemoglobin (Hb) repletion assay. Weanling male Wistar rats were fed a low-Fe diet (12 mg/kg) for 15 days followed by 2 weeks of Fe repletion with diets providing 35 mg Fe/kg as either ferrous sulfate (n = 8) or ferric pyrophosphate (FP; n = 12). At final day of each period (depletion and repletion), Fe-adequate animals were also euthanized. Iron status (blood Hb, Hb Fe pool, Hb regeneration efficiency), tibia mineral concentrations (Ca, Mg, Fe, Cu, and Zn) and biomechanical properties were evaluated. Iron-deficient rats had lower tibia Fe and Mg levels and bone strength when compared to controls. Yield load and resilience were positively related to tibia Mg levels (r = 0.47, P = 0.02 and r = 0.56, P = 0.004, respectively). Iron repletion did not recover tibia Mg concentrations impaired by Fe deficiency. Moreover, bone elastic properties were negatively affected by FP consumption. In conclusion, bone mineral composition and strength were affected by Fe deficiency, whereas dietary Fe source influenced tibia Mg and resistance in the period during which rats were recovering from anemia.     

The restoration of full-thickness cartilage defects with mesenchymal stem cells (MSCs) loaded and cross-linked bilayer collagen scaffolds on rabbit model

Cartilage has a limited self-repair capability and the repair of large cartilage defects remains a challenge in clinic. This study aimed to investigate the effect of mesenchymal stem cells (MSCs) loaded three-dimensional bilayer collagen scaffold for cartilage repair. Cross-linked three-dimensional bilayer collagen scaffolds seeded with or without MSCs were implanted into large cartilage defects (4 mm in diameter; 3 mm in depth) in rabbit knees. The untreated cartilage defects served as control. The tissue response was evaluated at 6 and 12 weeks after implantation by general histology and semi-quantitative histological grading systems. In addition, the repaired tissues were evaluated by mechanical test at 12 weeks after implantation. The MSCs-loaded collagen scaffold group showed the most hyaline cartilage, highest histological scores and compressive modulus. Moreover, it showed a good integration with the subchondral bone and adjacent cartilage. The structure of the novel bilayer collagen scaffolds provided architectural support for the differentiation of MSCs and demonstrated successful induction of in vivo chondrogenesis. These findings suggested that MSCs-loaded bilayer collagen scaffold could be an appealing candidate to be used for cartilage regeneration.     

Stromal cell derived factor-1α enhances bone formation based on in situ recruitment: a histologic and histometric study in rabbit calvaria

Histological methods were used to assess whether in situ recruitment using stromal cell derived factor-1α (SDF-1α) enhances bone formation. Four defects were created in the calvarias of 16 rabbits and filled with: (1) a blood clot only (group C); (2) autogenous bone particles (AB, 0.2 ml) (group AB); (3) AB (0.1 ml) + bone marrow derived stromal stem cells (group ABC); or (4) AB (0.1 ml) + SDF-1α (group ABS). Bone formation was significantly greater in groups AB and ABC compared with group ABS after 2 weeks (P < 0.05). Bone formation was similar between groups AB, ABC, and ABS after 4 weeks (P > 0.05). SDF-1α is a promising candidate for in situ recruitment in bone regeneration.     

Autologous mesenchymal stem cells loaded in Gelfoam<Superscript>®</Superscript> for structural bone allograft healing in rabbits

This study was designed to evaluate the effect of autologous bone marrow mesenchymal stem cells (MSCs) seeded into Gelfoam^® on structural bone allograft healing. Thirty New Zealand white rabbits were divided into two groups. Segmental bone defect was created on diaphysis of the femur, and the defect was reconstructed with structural bone allograft. In experimental group, structural allograft was wrapped around by Gelfoam^® containing autologous MSCs, whereas cells were not included in control group. At 4, 8, 12 weeks, the femur of rabbits underwent radiographic and histologic evaluation for bony union. Bone morphogenic protein-2 (BMP-2), BMP-4, BMP-7, vascular endothelial growth factor (VEGF), and receptor activator of nuclear factor-kappa B ligand (RANKL) were measured within the grafted periosteal tissue. Bony union was not achieved in both groups at 4 and 8 weeks. At 12 weeks, three out of five femurs in experimental group were united, but one out of five in control group was united. Mean Taira scores were significantly different between two groups. The expression of BMP-2 was significantly higher at 4, 8 weeks, the expressions of BMP-4 and BMP-7 were significantly higher at 8 and 12 weeks, and the expression of VEGF and RANKL were significantly higher at all time points in experimental group. Incorporation of the structural bone allograft could be enhanced if allograft is covered with Gelfoam^® containing autologous MSCs. MSCs have influence on not only bone formation, but neo-angiogenesis, and bone resorption.     

Suppression of the immune system as a critical step for bone formation from allogeneic osteoprogenitors implanted in rats

The surface marker profile of mesenchymal stromal cells (MSCs) suggests that they can escape detection by the immune system of an allogeneic host. This could be an optimal strategy for bone regeneration applications, where off‐the‐shelf cells could be implanted to heal bone defects. However, it is unknown how pre‐differentiation of MSCs to an osteogenic lineage, a means of improving bone formation, affects their immunogenicity. Using immunohistological techniques in a rat ectopic implantation model, we demonstrate that allogeneic osteoprogenitors mount a T cell‐ and B cell‐mediated immune response resulting in an absence of in vivo bone formation. Suppression of the host immune response with daily administration of an immunosuppressant, FK506, is effective in preventing the immune attack on the allogeneic osteoprogenitors. In the immunosuppressed environment, the allogeneic osteoprogenitors are capable of generating bone in amounts similar to those of syngeneic cells. However, using osteoprogenitors from one of the allogeneic donors led to newly deposited bone that was attacked by the host immune system, despite the continued administration of the immunosuppressant. This suggests that, although using an immunosuppressant can potentially suppress the immune attack on the allogeneic cells, optimizing the dose of the immunosuppressant may be crucial to ensure bone formation within the allogeneic environment. Overall, allografts comprising osteoprogenitors derived from allogeneic MSCs have the potential to be used in bone regeneration applications.     

Effects of raloxifene and estradiol on bone turnover parameters in intact and ovariectomized rats

This study was designed to investigate effects of raloxifene (RLX) and estradiol on bone formation and resorption in intact and ovariectomized (ovx) rat models. In the intact model, a total of 24 adult female rats were divided into three groups: Controls subcutaneously received saline alone. RLX (2 mg/kg) and estradiol (30 μg/kg) were injected to two groups of animals for a period of 6 weeks at two daily intervals. In the second model, rats (n = 24) were ovx and allowed to recover for a period of at least 3 weeks. Control group received vehicle alone. Remaining rats were divided into two groups and injected with RLX (2 mg/kg) and estradiol (30 μg/kg) for 6 weeks. Urine samples were collected from all animals 24 h after the last drug administration. Urinary deoxypyridinoline (DPD) was measured by ELISA. Serum parathyroid hormone (PTH), calcitonin, and osteocalcin levels were measured by immunoradiometric method. Serum concentrations of alkaline phosphatase (ALP), Ca, and inorganic phosphate were determined by enzymatic–colorimetric method. Lumbar vertebrae (L2) of all animals were dissected out and processed for histopathological evaluation. Removal of ovaries significantly elevated urinary DPD levels (p < 0.01) compared with intact controls. Treatment of both intact and ovx rats with estradiol resulted in significant decreases (p < 0.01) in DPD values. RLX administration had no significant effect in the intact rats, but it remarkably reduced bone turnover in the ovx animals (p < 0.001). Both estradiol and RLX produced conflicting effects on serum ALP, osteocalcin, and PTH levels in both animal models. These findings suggest that RLX exerts its protective effects by reducing bone resorption, similar to that of estradiol, in ovx rats.     

Transdifferentiated Mesenchymal Stem Cells as Alternative Therapy in Supporting Nerve Regeneration and Myelination

1. Aims: Demyelination plays a crucial role in neurodegenerative processes and traumatic disorders. One possibility to achieve remyelination and subsequent restoration of neuronal function is to provide an exogenous source of myelinating cells via transplantation. In this context, mesenchymal stem cells (MSCs) have attracted interest. They are multipotent stem cells that differentiate into cells of the mesodermal lineage like bone, cartilage, fat, and muscle. Although adult, their differentiation potential is remarkable, and they are able to transdifferentiate.2. Methods: We transformed cultivated rat MSCs into myelinating cells by using a cytokine cocktail. Transdifferentiated MSCs were characterized by an enhanced expression of LNGF-receptor, Krox20, and CD104, and a decreased expression of BMP receptor-1A as compared to untreated MSCs. The myelinating capacity was evaluated in vitro and in vivo. Therefore, PC12 cells, normally unmyelinated, were cocultivated with MSCs, transdifferentiated MSCs, and Schwann cells, or the respective cells were grafted into an autologous muscle conduit bridging a 2-cm gap in the rat sciatic nerve. Myelination of PC12 cells was demonstrated by electron microscopy. In vivo, after 3 and 6 weeks regeneration including myelination was monitored histologically and morphometrically. Autologous nerves and cell-free muscle grafts were used as control.3. Results: Schwann cells and transdifferentiated MSCs were able to myelinate PC12 cells after 14 days in vitro. In vivo, autologous nerve grafts demonstrated the best results in all regenerative parameters. An appropriate myelination was noted in the Schwann cell groups and, albeit with restrictions, in the transdifferentiated MSC groups, while regeneration in the MSC groups and in the cell-free groups was impaired.4. Conclusion: Our findings demonstrate that it may be possible to differentiate MSCs into therapeutically useful cells for clinical applications in myelin defects.     

Effect of guided bone regeneration on bone quality surrounding dental implants

Bone quality as well as its quantity at the implant interface is responsible for determining stability of the implant system. The objective of this study is to examine the nanoindentation based elastic modulus (E) at different bone regions adjacent to titanium dental implants with guided bone regeneration (GBR) treated with DBM and BMP-2 during different post-implantation periods. Six adult male beagle dogs were used to create circumferential defects with buccal bone removal at each implantation site of mandibles. The implant systems were randomly assigned to only GBR (control), GBR with demineralized bone matrix (DBM), and GBR with DBM + recombinant human bone morphogenetic protein-2 (rhBMP-2) (BMP) groups. Three animals were sacrificed at each 4 and 8 weeks of post-implantation healing periods. Following buccolingual dissection, the E values were assessed at the defects (Defect), interfacial bone tissue adjacent to the implant (Interface), and pre-existing bone tissue away from the implant (Pre-existing). The E values of BMP group had significantly higher than control and DBM groups for interface and defect regions at 4 weeks of post-implantation period and for the defect region at 8 weeks (p < 0.043). DBM group had higher E values than control group only for the defect region at 4 weeks (p < 0.001). The current results indicate that treatment of rhBMP-2 with GBR accelerates bone tissue mineralization for longer healing period because the GBR likely facilitates a microenvironment to provide more metabolites with open space of the defect region surrounding the implant.     

Allogeneic Mesenchymal Stem Cells in Combination with Hyaluronic Acid for the Treatment of Osteoarthritis in Rabbits

Mesenchymal stem cell (MSC)-based therapies may aid in the repair of articular cartilage defects. The purpose of this study was to investigate the effects of intraarticular injection of allogeneic MSCs in an in vivo anterior cruciate ligament transection (ACLT) model of osteoarthritis in rabbits. Allogeneic bone marrow-derived MSCs were isolated and cultured under hypoxia (1% O₂). After 8 weeks following ACLT, MSCs suspended in hyaluronic acid (HA) were injected into the knees, and the contralateral knees were injected with HA alone. Additional controls consisted of a sham operation group as well as an untreated osteoarthritis group. The tissues were analyzed by macroscopic examination as well as histologic and immunohistochemical methods at 6 and 12 weeks post-transplantation. At 6 and 12 weeks, the joint surface showed less cartilage loss and surface abrasion after MSC injection as compared to the tissues receiving HA injection alone. Significantly better histological scores and cartilage content were observed with the MSC transplantation. Furthermore, engraftment of allogenic MSCs were evident in surface cartilage. Thus, injection of the allogeneic MSCs reduced the progression of osteoarthritis in vivo.     

Microarchitecture and Nanomechanical Properties of Trabecular Bone After Strontium Administration in Osteoporotic Goats

Strontium (Sr) ralenate is a new agent used for the prevention and treatment of osteoporosis. As a bone-seeking element, 98% of Sr is deposited in the bone and teeth after oral ingestion. However, the effect of Sr treatment on bone microarchitecture and bone nanomechanical properties remains unclear. In this study, 18 osteoporotic goats were divided into four groups according to the treatment regimen: control, calcium alone (Ca), calcium and Sr at 24 mg/kg (Ca + 24Sr), and calcium and Sr at 40 mg/kg (Ca + 40Sr). The effects of Sr administration on bone microarchitecture and nanomechanical properties of trabecular bones were analyzed with micro-CT and nanoindentation test, respectively. Serum Sr levels increased six- and tenfold in the Ca + 24Sr and Ca + 40Sr groups, respectively. Similarly, Sr in the bone increased four- and sixfold in these two groups. Sr administration significantly increased trabecular bone volume fraction, trabecular thickness, and double-labeled new bone area. Sr administration, however, did not significantly change the nanomechanical properties of trabecular bone (elastic modulus and hardness). The data suggested that Sr administration increased trabecular bone volume and improved the microarchitecture while maintaining the intrinsic tissue properties in the osteoporotic goat model.     

The Effects of Chromium Histidinate on Mineral Status of Serum and Tissue in Fat-Fed and Streptozotocin-Treated Type II Diabetic Rats

The present study was conducted to investigate the effects of chromium histidinate (CrHis) against experimentally induced type II diabetes and on chromium (Cr), zinc (Zn), selenium (Se), manganese (Mn), iron (Fe), and copper (Cu) in serum, liver, and kidney of diabetic rats. The male Wistar rats (n = 60, 8 weeks old) were divided into four groups. Group I received a standard diet (12% of calories as fat); group II were fed standard diet and received CrHis (110 mcg CrHis/kg body weight per day); group III received a high-fat diet (HFD; 40% of calories as fat) for 2 weeks and then were injected with streptozotocin (STZ) on day 14 (STZ, 40 mg/kg i.p.; HFD/STZ); group IV were treated as group III (HFD/STZ) but supplemented with 110 mcg CrHis/kg body weight per day. The mineral concentrations in the serum and tissue were determined by atomic absorption spectrometry. Compared to the HFD/STZ group, CrHis significantly increased body weight and reduced blood glucose in diabetic rats (p < 0.001). Concentrations of Cr, Zn, Se, and Mn in serum, liver, and kidney of the diabetic rats were significantly lower than in the control rats (p < 0.0001). In contrast, higher Fe and Cu levels were found in serum and tissues from diabetic versus the non-diabetic rats (p < 0.001). Chromium histidinate supplementation increased serum, liver, and kidney concentrations of Cr and Zn both in diabetic and non-diabetic rats (p < 0.001). Chromium supplementation increased Mn and Se levels in diabetic rats (p < 0.001); however, it decreased Cu levels in STZ-treated group (p < 0.001). Chromium histidinate supplementation did not affect Fe levels in both groups (p > 0.05). The results of the present study conclude that supplementing Cr to the diet of diabetic rats influences serum and tissue Cr, Zn, Se, Mn, and Cu concentrations.