Ovariectomy of 12-month-old rats: effects on osteoprogenitor numbers in bone cell populations isolated from femur and on histomorphometric parameters of bone turnover in corresponding tibia

Ovariectomy (OVX) in rats results in increased bone turnover and decreased bone volume and bone mineral density when measured in the metaphyses of long bones. We have investigated the effects of OVX on changes in the number of progenitors in cell populations derived from the metaphyseal bone of femurs of ovariectomized rats at 12 months of age, by using colony assays, bone nodule assays, and limiting dilution analysis at 1.5 and 9 months post-OVX. We have also measured histomorphometric parameters of bone formation and resorption in the corresponding tibia at the same time-points. A significant increase, as shown by bone nodule assays and limiting dilution analysis, occurs in the number of progesterone- and dexamethasone-responsive osteoprogenitors in cell populations isolated from ovariectomized rats at the 9-month post-OVX time-point. Progesterone-responsive osteoprogenitors are also increased at 1.5 months post-OVX. The number of fibroblast colony-forming units does not change. Histomorphometry has shown that OVX causes an increase in osteoblast surfaces, mineralizing surfaces, and bone formation rate at both 1.5 and 9 months post-OVX. The mineral apposition rate is increased at 1.5 months post-OVX. OVX also increases parameters of bone resorption at both time-points, the net result being a decrease in bone mineral density and cancellous bone volume at 9 months post-OVX. Thus, OVX in rats at 12 months of age is associated with an increase in the number of both progesterone- and dexamethasone-responsive osteoprogenitors 9 months post-OVX; this corresponds with increases in the histomorphometric parameters of bone formation.     

Influence of fracture gap size on the pattern of long bone healing: a computational study

Following fractures, bones restore their original structural integrity through a complex process in which several cellular events are involved. Among other factors, this process is highly influenced by the mechanical environment of the fracture site. In this study, we present a mathematical model to simulate the effect of mechanical stimuli on most of the cellular processes that occur during fracture healing, namely proliferation, migration and differentiation. On the basis of these three processes, the model then simulates the evolution of geometry, distributions of cell types and elastic properties inside a healing fracture. The three processes were implemented in a Finite Element code as a combination of three coupled analysis stages: a biphasic, a diffusion and a thermoelastic step. We tested the mechano-biological regulatory model thus created by simulating the healing patterns of fractures with different gap sizes and different mechanical stimuli. The callus geometry, tissue differentiation patterns and fracture stiffness predicted by the model were similar to experimental observations for every analysed situation.     

Bone grafts,bone substitutes and orthobiologics: The bridge between basic science and clinical advancements in fracture healing

The biology of fracture healing is better understood than ever before, with advancements such as the locking screw leading to more predictable and less eventful osseous healing. However, at times one’s intrinsic biological response, and even concurrent surgical stabilization, is inadequate. In hopes of facilitating osseous union, bone grafts, bone substitutes and orthobiologics are being relied on more than ever before. The osteoinductive, osteoconductive and osteogenic properties of these substrates have been elucidated in the basic science literature and validated in clinical orthopaedic practice. Furthermore, an industry built around these items is more successful and in demand than ever before. This review provides a comprehensive overview of the basic science, clinical utility and economics of bone grafts, bone substitutes and orthobiologics.     

Primary cortical brain cells influence osteoblast activity

The presence of neuropeptides and neuroreceptors in the bone have been reported in several studies. Bone turn-over seems to be controlled by the nervous system. The actual pathway or the control mechanism is still under investigation. In this study we investigate the changes in osteoblast cells if they are in co-culture with primary cortical brain cells. After seven days in co-culture with the primary fetal brain cells the osteoblast cells exhibited hypertrophic morphological changes and showed stronger ALP activity.     

Modulation of bone morphogenetic protein antagonists to stimulate clinical osteogenesis

Bone morphogenetic proteins (BMPs) are important for the development and functioning of a wide variety of tissues and organ systems. Their ability to induce bone formation has been harnessed for clinical application. Specifically, local application of BMPs into fractures and fusions has shown some efficacy in inducing bone formation. However, clinical success has not been as robust as might be expected from the results obtained using animal models. This difference may be due to a number of mechanisms regulating BMP activity in vivo. One class of major regulators is the extracellular antagonist (e.g. Noggin, Gremlin, DAN), the dysfunction of which has been shown to result in ectopic bone formation in animal models and human disease. We hypothesize that local application of BMPs at high concentrations induces increased production of BMP antagonists, thereby limiting BMP activity and clinical efficacy. Therapies blocking the function of BMP antagonists should therefore result in enhanced BMP activity and increased bone formation. Furthermore, titrated systemic regulation of BMP antagonist may potentially reverse osteoporosis. Our collective experience with the clinical use of BMP illustrates the importance of understanding mechanisms of endogenous antagonism and regulation in the exogenous application of a protein as a therapeutic.     

Effects of the addition of vancomycin on the physical and handling properties of calcium sulfate bone cement

Intra-operative applications of bone graft substitutes into bone voids support mechanical stability, and accelerate fracture healing. Calcium sulfate bone cement, an injectable substitute, is used widely in non-loading bones with favorable results. In addition, calcium sulfate also serves as a vehicle for antibiotics that treat osteomyelitis or prevent contaminations. However, the effects of the addition of antibiotics on the physical properties of calcium sulfate are rarely addressed. In this study, calcium sulfates mixed with vancomycin at different weight ratios (4:0, 4:0.025, 4:0.05, 4:0.075, and 4:0.1) were evaluated in vitro. No obvious temperature increase or pH change was observed during setting and immersion in the simulated body fluid. The added vancomycin did not influence the mechanical strength, crystalline phase, or microstructure of the calcium sulfate cement. However, the addition of vancomycin extended the initial and final setting time (4:0.075, and 4:0.1). A higher amount of vancomycin resulted in a higher initial boosting release, but did not lead to faster degradation. The vancomycin-impregnated cement exhibited inhibitory effects against Staphylococcus aureus. These data indicate that the extended initial and final setting time of the calcium sulfate bone cement with the addition of vancomycin should be considered during operation.     

Segmental bone replacement via patient‐specific,three‐dimensional printed bioresorbable graft substitutes and their use as templates for the culture of mesenchymal stem cells under mechanical stimulation at various frequencies

The treatment of large segmental bone defects remains a challenge as infection, delayed union, and nonunion are common postoperative complications. A three‐dimensional printed bioresorbable and physiologically load‐sustaining graft substitute was developed to mimic native bone tissue for segmental bone repair. Fabricated from polylactic acid, this graft substitute is novel as it is readily customizable to accommodate the particular size and location of the segmental bone of the patient to be replaced. Inspired by the structure of the native bone tissue, the graft substitute exhibits a gradient in porosity and pore size in the radial direction and exhibit mechanical properties similar to those of the native bone tissue. The graft substitute can serve as a template for tissue constructs via seeding with stem cells. The biocompatibility of such templates was tested under in vitro conditions using a dynamic culture of human mesenchymal stem cells. The effects of the mechanical loading of cell‐seeded templates under in vitro conditions were assessed via subjecting the tissue constructs to 28 days of daily mechanical stimulation. The frequency of loading was found to have a significant effect on the rate of mineralization, as the alkaline phosphatase activity and calcium deposition were determined to be particularly high at the typical walking frequency of 2 Hz, suggesting that mechanical stimulation plays a significant role in facilitating the healing process of bone defects. Utilization of such patient‐specific and biocompatible graft substitutes, coupled with patient’s bone marrow cells seeded and exposed to mechanical stimulation of 2 Hz have the potential of reducing significant volumes of cadaveric tissue required, improving long‐term graft stability and incorporation, and alleviating financial burdens associated with delayed or failed fusions of long bone defects.     

Temporal sequence of interleukin 1α—mediated stimulation and inhibition of bone formation by isolated fetal rat calvaria cells in vitro

Cytokines released at sites of inflammation and infection may alter normal bone remodeling processes resulting in pathologic bone destruction or bone formation. Interleukin 1, an inflammatory mediator, has been shown to stimulate as well as inhibit parameters associated with bone formation. In this study we have examined temporal aspects of the biphasic effects of recombinant interleukin 1 alpha (IL 1 alpha) on the differentiation of osteoprogenitor cells into bone-forming osteoblasts (bone nodules) in vitro. A dose-dependent stimulation of bone formation over a concentration range of 0.5 to 50 U/mL (1.4 x 10(-12) to 1.4 x 10(-10) M) was observed when preconfluent, primary cultures of fetal rat calvaria (RC) cells were pulsed with IL 1 alpha for 72 to 96 hr from the beginning of the culture period. This was correlated with a stimulation of cell proliferation and alkaline phosphatase activity measured during the late log phase of growth. In contrast, continuous exposure to IL 1 alpha or exposure to IL 1 alpha after confluency resulted in inhibition of bone nodule formation and alkaline phosphatase activity. IL 1 alpha-stimulated prostaglandin E2 (PGE2) production until the RC cells became multilayered, but the addition of the cyclooxygenase inhibitor indomethacin had no effect in reducing the IL 1 alpha-mediated stimulation of cell proliferation or bone nodule formation. However, in cultures continuously exposed to IL 1 alpha, added indomethacin partially reduced the inhibition of bone formation, suggesting that prostaglandin production may play a role in the inhibitory effects of IL 1 alpha on bone formation.     

Ability of a bovine bone graft, alone or enriched with PDGF-BB or rhBMP-2, to promote human periodontal ligament (PDL) cells proliferation. A preliminary study

One of the most important goals of the periodontal therapy procedures is to stimulate the formation of new bone into osseous defects resulted from periodontal disease. A wide range of grafting materials is used to achieve this aim. Recently, the Human Tissue Bank of the National Center for Scientific Research Demokritos in Athens (Greece) has prepared, in a preliminary study, a cancellous bovine-derived bone matrix (BBM). The purpose of the present work was to investigate the role of this bovine bone material in the periodontal regeneration, by studying the rate of human periodontal ligament (PDL) cells proliferation in the presence of this matrix alone, or after the addition of the growth factors, platelet-derived growth factor-BB (PDGF-BB) or recombinant human bone morphogenetic protein-2 (rhBMP-2).Bovine bone graft was prepared using the know how acquired by the 30 years continuous preparation and delivery of lyophilized human bone grafts by the Demokritos Bank.PDL cells cultures were derived from the mid root of two maxillary premolars. The teeth were caries-free and were extracted for orthodontic reasons from 1 adult female patient. Cells were grown in 24-well dishes in the presence of 20 mg BBM. On day 2 of quiescence, new medium was added with 10 ng/ml of PDGF-BB or 50 ng/ml of rhBMP-2. To determine the effects of the test agents on cell proliferation, DNA synthesis was estimated by measuring [³H] thymidine incorporation. After 48 h of incubation the cells were processed to subject to scintillation counting. Counts per minute (cpm/well) were determined for each sample.The results revealed that this BBM has the ability to maintain PDL cells proliferation and could be used as an alternative graft material. PDGF-BB when added improved the cell proliferative response resulting in a more active BBM, while the presence of rhBMP-2 did not support cell mitosis.     

Dysfunctional stem and progenitor cells impair fracture healing with age

Successful fracture healing requires the simultaneous regeneration of both the bone and vasculature; mesenchymal stem cells(MSCs) are directed to replace the bone tissue, while endothelial progenitor cells(EPCs) form the new vasculature that supplies blood to the fracture site. In the elderly, the healing process is slowed, partly due to decreased regenerative function of these stem and progenitor cells. MSCs from older individuals are impaired with regard to cell number, proliferative capacity, ability to migrate, and osteochondrogenic differentiation potential. The proliferation, migration and function of EPCs are also compromised with advanced age. Although the reasons for cellular dysfunction with age are complex and multidimensional, reduced expression of growth factors, accumulation of oxidative damage from reactive oxygen species,and altered signaling of the Sirtuin-1 pathway are contributing factors to aging at the cellular level of both MSCs and EPCs. Because of these geriatric-specific issues, effective treatment for fracture repair may require new therapeutic techniques to restore cellular function. Some suggested directions for potential treatments include cellular therapies, pharmacological agents, treatments targeting age-related molecular mechanisms, and physical therapeutics.Advanced age is the primary risk factor for a fracture, due to the low bone mass and inferior bone quality associated with aging; a better understanding of the dysfunctional behavior of the aging cell will provide a foundation for new treatments to decrease healing time and reduce the development of complications during the extended recovery from fracture healing in the elderly.     

Adipose tissue in bone regeneration - stem cell source and beyond

Adipose tissue (AT) is recognized as a complex organ involved in major home-ostatic body functions, such as food intake, energy balance, immunomodulation, development and growth, and functioning of the reproductive organs. The role of AT in tissue and organ homeostasis, repair and regeneration is increasingly recognized. Different AT compartments (white AT, brown AT and bone marrow AT) and their interrelation with bone metabolism will be presented. AT-derived stem cell populations - adipose-derived mesenchymal stem cells and pluripotent-like stem cells. Multilineage differentiating stress-enduring and dedifferentiated fat cells can be obtained in relatively high quantities compared to other sources. Their role in different strategies of bone and fracture healing tissue engineering and cell therapy will be described. The current use of AT- or AT-derived stem cell populations for fracture healing and bone regenerative strategies will be presented, as well as major challenges in furthering bone regenerative strategies to clinical settings.     

Antler stem cells and their potential in wound healing and bone regeneration

Compared to other vertebrates, the regenerative capacity of appendages in mammals is very limited. Deer antlers are an exception and can fully regenerate annually in postnatal mammals. This process is initiated by the antler stem cells (AnSCs). AnSCs can be divided into three types: (1) Antlerogenic periosteum cells (for initial pedicle and first antler formation); (2) Pedicle periosteum cells (for annual antler regeneration); and (3) Reserve mesenchyme cells (RMCs) (for rapid antler growth). Previous studies have demonstrated that AnSCs express both classic mesenchymal stem cells (MSCs) and embryonic stem cells (ESCs), and are able to differentiate into multiple cell types in vitro. Thus, AnSCs were defined as MSCs, but with partial ESC attributes. Near-perfect generative wound healing can naturally occur in deer, and wound healing can be achieved by the direct injection of AnSCs or topical application of conditioned medium of AnSCs in rats. In addition, in rabbits, the use of both implants with AnSCs and cell-free preparations derived from AnSCs can stimulate osteogenesis and repair defects of bone. A more comprehensive understanding of AnSCs will lay the foundation for developing an effective clinical therapy for wound healing and bone repair.     

Reconstruction of radial bone defect using gelatin sponge and a BMP-2 combination graft

Many bioactive molecules like recombinant human bone morphogenetic protein 2 (rhBMP-2) have been developed for mineralized bone grafts, for which proper scaffolds are necessary to successfully apply the bioactive molecules. In this study, we tested the osteogenic efficacy of rhBMP-2 produced in-house in combination with gelatin sponge as the scaffold carrier in a rabbit radial defect model. The efficacy of the rhBMP-2 was determined by alkaline phosphatase activity assay of C2C12 cells. Two groups of ten rabbits each were treated with rhBMP-2/gelatin sponge, or gelatin sponge only. At 4 weeks, rhBMP-2/gelatin sponge grafts showed more bone regeneration than gelatin sponge grafts, as determined by X-ray radiography, micro-computed tomography, and histological analyses. At 8 weeks, rhBMP-2/gelatin sponge grafts exerted much stronger osteogenic effects. The study demonstrates the improved osteogenic efficacy of the rhBMP-2/gelatin sponge grafts in a rabbit radial bone defect model acting as a bone-inductive material. [BMB Reports 2013; 46(6): 328-333]     

Mechanical properties of compacted morselized cancellous bone graft using one-dimensional consolidation testing

Failures of orthopaedic procedures that use morselized cancellous bone (MCB) graft for load bearing are often due to gross displacement within the graft material. For this reason the mechanical behavior of MCB must be better understood. Our purpose is to present a detailed testing methodology for the mechanical characterization of MCB, and to illustrate how this methodology can be used to study the influence of water and fat content. Complete one-dimensional consolidation testing was performed on bovine cancellous bone processed to represent MCB typically used in surgery (52% water, 31% fat). The one-dimensional consolidation strain under a stress of 1.09 MPa was 30.9% and the confined modulus was 8.0 MPa. The coefficient of consolidation (rate of consolidation) was 2.2×10⁻⁵ cm²/s and the coefficient of secondary strain (steady-state creep rate) was 1.9%. While reducing the water content alone had some influence on properties, reducing the fat content improved both the static and dynamic behavior. A sample of MCB which had fat intentionally minimized and a lower overall moisture content (56% water, 5% fat) demonstrated 23.1% strain, a confined modulus of 9.6 MPa, a coefficient of consolidation of 3.4×10⁻³ cm²/s, and a coefficient of secondary strain of 0.9%. The test methods described in this technical note can be used to evaluate the influence of fluid content on the mechanical behavior of MCB.     

In vitro evaluation of alginate encapsulated adipose-tissue stromal cells for use as injectable bone graft substitute

This study aims to investigate the survival and osteogenic behavior of murine-derived adipose-tissue stromal cells (ATSCs) encapsulated in alginate microcapsules thereby instigating further studies in this cell delivery strategy for in vivo osteogenesis. Cell viability was quantified using a tetrazolium-based assay and osteogenic differentiation was evaluated by both alkaline-phosphatase (ALP) histochemistry and osteocalcin mRNA analysis. Following microencapsulation, cell numbers increased from 3.9 x 10(3) on day 1 to 7.8 x 10(3) on day 7 and maintained excellent viability in the course of 21-day culture. ALP was 6.9, 5.5, and 3.2 times higher than monolayer cultures on days 7, 14, and 21, respectively. In addition, osteocalcin mRNA was detectable in encapsulated cultures earlier (day 14) than monolayer cultures. We conclude that alginate microcapsules can act as three-dimensional matrix for ATSC proliferation and has potential for use as injectable, biodegradable scaffold in bone tissue engineering.     

Dicer inactivation in osteoprogenitor cells compromises fetal survival and bone formation, while excision in differentiated osteoblasts increases bone mass in the adult mouse

MicroRNA attenuation of protein translation has emerged as an important regulator of mesenchymal cell differentiation into the osteoblast lineage. A compelling question is the extent to which miR biogenesis is obligatory for bone formation. Here we show conditional deletion of the Dicer enzyme in osteoprogenitors by Col1a1-Cre compromised fetal survival after E14.5. A mechanism was associated with the post-commitment stage of osteoblastogenesis, demonstrated by impaired ECM mineralization and reduced expression of mature osteoblast markers during differentiation of mesenchymal cells of ex vivo deleted Dicer^c/c. In contrast, in vivo excision of Dicer by Osteocalcin-Cre in mature osteoblasts generated a viable mouse with a perinatal phenotype of delayed bone mineralization which was resolved by 1 month. However, a second phenotype of significantly increased bone mass developed by 2 months, which continued up to 8 months in long bones and vertebrae, but not calvariae. Cortical bone width and trabecular thickness in Dicer^Δoc/Δoc was twice that of Dicer^c/c controls. Normal cell and tissue organization was observed. Expression of osteoblast and osteoclast markers demonstrated increased coupled activity of both cell types. We propose that Dicer generated miRs are essential for two periods of bone formation, to promote osteoblast differentiation before birth, and control bone accrual in the adult.     

骨髓间充质干细胞向神经细胞分化的研究

无论是在体外实验、还是在体内实验,MSCs都可以向中枢神经系统(CNS)神经细胞分化,但争议颇多。因为功能性神经元不仅要具有典型神经元的形态、特异性标记,还要求具有可兴奋性、能和其他神经元形成突触联系、产生突触电位等,所以对于骨髓间充质干细胞是否能诱导出真正具有功能的神经元存在很大分歧。在此对MSCs向神经细胞诱导分化研究的现况、存在的问题及发展前景给以综述。