Direct Osteogenesis Research Articles

A distinct "repair" role of regulatory T cells in fracture healing.

Regulatory T cells (Tregs) suppress immune responses and inflammation. Here, we described the distinct nonimmunological role of Tregs in fracture healing. The recruitment from the circulation pool, peripheral induction, and local expansion rapidly enriched Tregs in the injured bone. The Tregs in the injured bone displayed superiority in direct osteogenesis over Tregs from lymphoid organs. Punctual depletion of Tregs compromised the fracture healing process, which leads to increased bone nonunion. In addition, bone callus Tregs showed unique T-cell receptor repertoires. Amphiregulin was the most overexpressed protein in bone callus Tregs, and it can directly facilitate the proliferation and differentiation of osteogenic precursor cells by activation of phosphatidylinositol 3-kinase/protein kinase B signaling pathways. The results of loss- and gain-function studies further evidenced that amphiregulin can reverse the compromised healing caused by Treg dysfunction. Tregs also enriched in patient bone callus and amphiregulin can promote the osteogenesis of human pre-osteoblastic cells. Our findings indicate the distinct and nonredundant role of Tregs in fracture healing, which will provide a new therapeutic target and strategy in the clinical treatment of fractures.

EFFECT OF USING MULTIPOTENT MESENCHYMAL STROMAL CELLS IN BONE GRAFTING WITH XENOGENIC BIOMATERIALS

Currently, there is a significant frequency of degenerative diseases of the spine after surgical interventions with the use of bone grafting, especially in elderly patients. The results of the research indicate that the use of stem cells is one of the promising areas to increase the efficiency of osseointegration. The aim of the study was to evaluate the effectiveness of osteogenesis in the conditions of using xenografts loaded with stem cells, as well as morphological features of osseointegration. Materials and methods. An experimental study was conducted on 22 male rabbits. 2 animals were used to obtain stem cells, the remaining animals were implanted with xenogenic bone–substituting material "Osteomatrix" in the iliac wing, of which 10 animals were implanted with a xenograft not populated with cells, and the other 10 animals were implanted with an identical xenograft populated with mesenchymal stromal cells. Withdrawal from the experiment was carried out on the 60th day after implantation. The material's examination was carried out using the methods of fluorescence and light microscopy. To assess the statistical significance of observed differences (evaluation of proliferation, neoangiogenesis and osseointegration) in the experimental and control groups, the Mann–Whitney U test was used with a critical significance of p ≤ 0.05. Results. When using an osteoplastic material that was not loaded with multipotent mesenchymal stromal cells within 60 days after surgery, osseointegration passes through indirect osteogenesis with formation of full-fledged bone tissue in the future, which increases the time of complete defect healing. In the case of using an osteomatrix loaded with multipotent mesenchymal stromal cells of the bone marrow, osteogenesis proceeds in a direct way with the formation of a full-fledged bone tissue. Osteomatrix loading with multipotent mesenchymal stromal cells stimulates neoangiogenesis and proliferative activity of the tissue, which promotes activation of bone tissue repair processes and stimulates the processes of xenograft osseointegration. Conclusions. The use of multipotent mesenchymal stromal cells in bone grafting using xenografts increases the efficiency of osseointegration by stimulating direct osteogenesis, increasing the activity of proliferation and angiogenesis.

Effect of Hydroxyapatite/β-Tricalcium Phosphate on Osseointegration after Implantation into Mouse Maxilla

In our previous study we established an animal model for immediately placed implants using mice and clarified that there were no significant differences in the chronological healing process at the bone-implant interface between immediately and delayed placed implants blasted with hydroxyapatite (HA)/β-tricalcium phosphate (β-TCP) (ratio 1:4). This study aimed to analyze the effects of HA/β-TCP on osseointegration at the bone-implant interface after immediately placed implants in the maxillae of 4-week-old mice. Right maxillary first molars were extracted and cavities were prepared with a drill and titanium implants, blasted with or without HA/β-TCP, were placed. The fixation was followed-up at 1, 5, 7, 14, and 28 days after implantation, and the decalcified samples were embedded in paraffin and prepared sections were processed for immunohistochemistry using anti-osteopontin (OPN) and Ki67 antibodies, and tartrate-resistant acid phosphatase histochemistry. The undecalcified sample elements were quantitatively analyzed by an electron probe microanalyzer. Bone formation occurred on the preexisting bone surface (indirect osteogenesis) and on the implant surface (direct osteogenesis), indicating that osseointegration was achieved until 4 weeks post-operation in both of the groups. In the non-blasted group, the OPN immunoreactivity at the bone-implant interface was significantly decreased compared with the blasted group at week 2 and 4, as well as the rate of direct osteogenesis at week 4. These results suggest that the lack of HA/β-TCP on the implant surface affects the OPN immunoreactivity on the bone-implant interface, resulting in decreased direct osteogenesis following immediately placed titanium implants.

MORPHOLOGICAL CHARACTERISTICS OF REPARATIVE OSTEOGENESIS IN THE RATS LOWER JAW UNDER THE CONDITIONS OF USING ELECTRICAL STIMULATION.

Aim: The purpose of the study was to identify the morphological features of reparative osteogenesis in the rats lower jaw under the conditions of using electrical stimulation. Materials and Methods: An experiment was conducted on 24 mature male rats of the WAG population. Two groups were formed. Group 1 included 12 rats that were modeled with a perforated defect of the lower jaw body. Group 2 included 12 animals that were modeled with a perforated defect similar to group 1. In animals, a microdevice for electrical action was implanted subcutaneously in the neck area on the side of the simulated bone defect (a temporary Videx AG 4 battery; a constant sinusoidal electric current of an unchanging nature 1 milliampere, frequency 30 W). The negative electrode connected to the negative pole of the battery was in contact with the bone defect. The battery and electrode were insulated with plastic heat shrink material. Morphological and statistical methods were used. Results: The positive effect of electrical stimulation on reparative osteogenesis was due to a decrease in the severity of hemodynamic disorders, activation of angiogenesis in granulation tissue, which was one of the components of the regenerate that filled the bone defect, matured and turned into connective tissue; stimulation of the proliferative potential of fibroblastic cells and cells with osteoblastic activity in granulation tissue; increasing the proliferative potential of osteoblastic elements of bone tissue bordering the cavity; stimulation of macrophage cells and processes of cleansing the bone cavity from fragments of a blood clot and alteratively changed tissues; formation of clusters of adipocytes in the loci of connective and granulation tissue of the regenerate; the process of metaplasia of connective tissue into bone tissue; an increase of the foci of hematopoiesis in the intertrabecular spaces of lamellar bone tissue. Conclusions: A comprehensive clinical and experimental study conducted by the authors proved that electrical stimulation activates the reparative osteogenesis in the lower jaw, which occurs through direct osteogenesis and does not finish on the 28th day of the experiment.

Direct osteogenesis and immunomodulation dual function via sustained release of naringin from the polymer scaffold.

Many traditional Chinese medicine monomers, such as naringin (NG), can regulate the local immune microenvironment to benefit osteogenesis. However, the rapid release of NG from scaffolds severely influences the osteogenesis-promoting effect. Herein, NG was loaded into mesoporous bioglass (MBG) to achieve sustained release through physical adsorption and the barrier role of mesoporous channels, then MBG loaded with NG was added to poly(L-lactic acid) (PLLA) to fabricate composite scaffolds by selective laser sintering (SLS) technology. The results showed that the NG-MBG/PLLA scaffolds could continuously and slowly release NG for 14 days compared with NG/PLLA scaffolds, and the cumulative release amount for the NG-MBG/PLLA scaffolds was 44.26%. In addition, the NG-MBG/PLLA scaffolds can promote the proliferation and osteogenesis differentiation of mouse bone marrow mesenchymal stem cells (mBMSCs). Meanwhile, the composite scaffolds decreased the reactive oxygen species (ROS) level of RAW264.7 under the stimulation of lipopolysaccharide (LPS) and significantly suppressed interleukin-6 (IL-6) and enhanced arginase-1 (Arg-1) protein expressions. Moreover, calcium nodule and alkaline phosphatase production of mBMSCs in a macrophage-conditioned medium for the NG-MBG/PLLA group also evidently increased compared with the PLLA and MBG/PLLA groups. These NG sustained-release composite scaffolds with osteo-immunomodulation function have great application prospects in the clinic.

Piezoresistive MXene/Silk fibroin nanocomposite hydrogel for accelerating bone regeneration by Re-establishing electrical microenvironment.

The electrical microenvironment plays an important role in bone repair. However, the underlying mechanism by which electrical stimulation (ES) promotes bone regeneration remains unclear, limiting the design of bone microenvironment–specific electroactive materials. Herein, by simple co-incubation in aqueous suspensions at physiological temperatures, biocompatible regenerated silk fibroin (RSF) is found to assemble into nanofibrils with a β-sheet structure on MXene nanosheets, which has been reported to inhibit the restacking and oxidation of MXene. An electroactive hydrogel based on RSF and bioencapsulated MXene is thus prepared to promote efficient bone regeneration. This MXene/RSF hydrogel also acts as a piezoresistive pressure transducer, which can potentially be utilized to monitor the electrophysiological microenvironment. RNA sequencing is performed to explore the underlying mechanisms, which can activate Ca2+/CALM signaling in favor of the direct osteogenesis process. ES is found to facilitate indirect osteogenesis by promoting the polarization of M2 macrophages, as well as stimulating the neogenesis and migration of endotheliocytes. Consistent improvements in bone regeneration and angiogenesis are observed with MXene/RSF hydrogels under ES in vivo. Collectively, the MXene/RSF hydrogel provides a distinctive and promising strategy for promoting direct osteogenesis, regulating immune microenvironment and neovascularization under ES, leading to re-establish electrical microenvironment for bone regeneration.

Regulating Macrophage Polarization in High Glucose Microenvironment Using Lithium-Modified Bioglass-Hydrogel for Diabetic Bone Regeneration.

Diabetes mellitus is a chronic metabolic disease with a proinflammatory microenvironment, causing poor vascularization and bone regeneration. Due to the lack of effective therapy and one-sided focus on the direct angiogenic properties of biomaterials and osteogenesis stimulation, the treatment of diabetic bone defect remains challenging and complex. In this study, using gelatin methacryloyl (GelMA) as a template, a lithium (Li) -modified bioglass-hydrogel for diabetic bone regeneration is developed. It exhibits a sustained ion release for better bone regeneration under diabetic microenvironment. The hydrogel is shown to be mechanically adaptable to the complex shape of the defect. In vitro, Li-modified bioglass-hydrogel promoted cell proliferation, direct osteogenesis, and regulated macrophages in high glucose (HG) microenvironment, with the secretion of bone morphogenetic protein-2 and vascular endothelial growth factor to stimulate osteogenesis and neovascularization indirectly. In vivo, composite hydrogels containing GelMA and Li-MBG (GM/M-Li) release Li ions to relieve inflammation, providing an anti-inflammatory microenvironment for osteogenesis and angiogenesis. Applying Li-modified bioglass-hydrogel, significantly enhances bone regeneration in a diabetic rat bone defect. Together, both remarkable in vitro and in vivo outcomes in this study present an opportunity for diabetic bone regeneration on the basis of HG microenvironment.

Advances in porous inorganic nanomaterials for bone regeneration

Porous inorganic materials such as mesoporous silica nanoparticles (MSNs), mesoporous bioactive glasses (MBGs), porous calcium phosphates, and metal–organic frameworks (MOFs) are used for bone regeneration due to their osteoinductive and porous properties. The direct osteogenesis ability can be adjusted by the design and composition of those inorganic materials. With porous structure, adjustable pore size and high surface area, they are used as carriers to deliver various small molecular drugs, proteins, and genes locally to promote bone generation. The surface of those porous inorganic materials can be further functionalized to control the loading and release of drugs and modulate the behaviour of host cells. This review summarizes the recent advances of various porous inorganic nanomaterials for bone repairing with a focus on their performance as scaffolds and drug delivery systems. We also discuss the challenges and prospects of porous inorganic nanomaterials for the future clinical application for bone regeneration.

IMPLANT DESIGN FACTORS THAT AFFECT PRIMARY STABILITY AND OSSEOINTEGRATION

Osseointegration can be defined as a direct structural and functional link between bone and implant surface. Asymptomatic and rigid fixation of alloplastic material in bone is crucial for implant stability and longterm success of dental implant treatment. The connection between implant and surrounding tissue is extremely dynamic. This interaction is the basis for the formation of a new structure - implant-tissue interface. The process of osseointegration is a combination of biological mechanisms as a result of which bone is formed on the walls of the osteotomous lodge growing to the implant surface (remote osteogenesis) and direct osteogenesis of bone on the surface of the implant (contact osteogenesis). The process is complex and there are a number of factors that affect the formation and maintenance of bone in the peri-implant space. The aim of the present study is to review the literature data on the macroscopic implant factors that affect primary stability and osseointegration

Osteopontin on the Dental Implant Surface Promotes Direct Osteogenesis in Osseointegration.

After dental implantation, osteopontin (OPN) is deposited on the hydroxyapatite (HA) blasted implant surface followed by direct osteogenesis, which is significantly disturbed in Opn-knockout (KO) mice. However, whether applying OPN on the implant surface promotes direct osteogenesis remains unclarified. This study analyzed the effects of various OPN modified protein/peptides coatings on the healing patterns of the bone-implant interface after immediately placed implantation in the maxilla of four-week-old Opn-KO and wild-type (WT) mice (n = 96). The decalcified samples were processed for immunohistochemistry for OPN and Ki67 and tartrate-resistant acid phosphatase histochemistry. In the WT mice, the proliferative activity in the HA binding peptide-OPN mimic peptide fusion coated group was significantly higher than that in the control group from day 3 to week 1, and the rates of OPN deposition and direct osteogenesis around the implant surface significantly increased in the recombinant-mouse-OPN (rOPN) group compared to the Gly-Arg-Gly-Asp-Ser peptide group in week 2. The rOPN group achieved the same rates of direct osteogenesis and osseointegration as those in the control group in a half period (week 2). None of the implant surfaces could rescue the direct osteogenesis in the healing process in the Opn-KO mice. These results suggest that the rOPN coated implant enhances direct osteogenesis during osseointegration following implantation.

Metformin-Incorporated Gelatin/Nano-Hydroxyapatite Scaffolds Promotes Bone Regeneration in Critical Size Rat Alveolar Bone Defect Model.

In this study, we fabricated gelatin/nano-hydroxyapatite/metformin scaffold (GHMS) and compared its effectiveness in bone regeneration with extraction-only, Sinbone, and Bio-Oss Collagen® groups in a critical size rat alveolar bone defect model. GHMS was synthesized by co-precipitating calcium hydroxide and orthophosphoric acid within gelatin solution, incorporating metformin, and cross-linked by microbial transglutaminase. The morphology, characterization, and biocompatibility of scaffold were examined. The in vitro effects of GHMS on osteogenic gene and protein expressions were evaluated. In vivo bone formation was assessed in a critical size rat alveolar bone defect model with micro-computed tomography and histological examination by comparing GHMS with extraction-only, Sinbone, and Bio-Oss Collagen®. The synthesized GHMS had a highly interconnected porous structure with a mean pore size of 81.85 ± 13.8 µm. GHMS exhibited good biocompatibility; promoted ALPL, RUNX2, SP7, BGLAP, SPARC and Col1a1 gene expressions; and upregulated the synthesis of osteogenic proteins, including osteonectin, osteocalcin, and collagen type I. In critical size rat alveolar bone defects, GHMS showed superior bone regeneration compared to extraction-only, Sinbone, and Bio-Oss Collagen® groups as manifested by greater alveolar ridge preservation, while more bone formation with a lower percentage of connective tissue and residual scaffold at the defect sites grafted with GHMS in histological staining. The GHMS presented in this study may be used as a potential bone substitute to regenerate alveolar bone. The good biocompatibility, relatively fast degradation, interconnected pores allowing vascularization, and higher bioactivity properties of the components of the GHMS (gelatin, nHA, and metformin) may contribute to direct osteogenesis.

Impairment of type H vessels by NOX2-mediated endothelial oxidative stress: critical mechanisms and therapeutic targets for bone fragility in streptozotocin-induced type 1 diabetic mice.

Rationale: Mechanisms underlying the compromised bone formation in type 1 diabetes mellitus (T1DM), which causes bone fragility and frequent fractures, remain poorly understood. Recent advances in organ-specific vascular endothelial cells (ECs) identify type H blood vessel injury in the bone, which actively direct osteogenesis, as a possible player.Methods: T1DM was induced in mice by streptozotocin (STZ) injection in two severity degrees. Bony endothelium, the coupling of angiogenesis and osteogenesis, and bone mass quality were evaluated. Insulin, antioxidants, and NADPH oxidase (NOX) inhibitors were administered to diabetic animals to investigate possible mechanisms and design therapeutic strategies.Results: T1DM in mice led to the holistic abnormality of the vascular system in the bone, especially type H vessels, resulting in the uncoupling of angiogenesis and osteogenesis and inhibition of bone formation. The severity of osteopathy was positively related to glycemic levels. These pathological changes were attenuated by early-started, but not late-started, insulin therapy. ECs in diabetic bones showed significantly higher levels of reactive oxygen species (ROS) and NOX 1 and 2. Impairments of bone vessels and bone mass were effectively ameliorated by treatment with anti-oxidants or NOX2 inhibitors, but not by a NOX1/4 inhibitor. GSK2795039 (GSK), a NOX2 inhibitor, significantly supplemented the insulin effect on the diabetic bone.Conclusions: Diabetic osteopathy could be a chronic microvascular complication of T1DM. The impairment of type H vessels by NOX2-mediated endothelial oxidative stress might be an important contributor that can serve as a therapeutic target for T1DM-induced osteopathy.

Pilot study of bone tissue reparative regeneration of the young Trachemys scripta shell

The turtles skeleton plan is an evolutionary developmental novelty in connection with the formation of a tortoise-specific carapacial ridge, which induces the growth of ribs in the dorsal direction and the subsequent formation of several carapace plates. The bone plates of these reptiles have different histogenesis: the neural and costal plates develop according to the mechanism of indirect osteogenesis by perichondral ossification of the axial skeleton elements (vertebrae and ribs), the peripheral and plastron bones develop according to the mechanism of direct osteogenesis in the dermis of the skin - these are the so-called «skin bones», which are derived from individual osteogenic cells outside the axial skeleton and develop by intramembranous osteogenesis. An experiment was carried out to study the post-traumatic osteogenesis of the carapace bone plates on a freshwater turtles Trachemys scripta at the age of 3 months. Two defects of carapace costal plates with a diameter of 4 mm and a depth to the fascia were performed with further routine histological examination. It was found that already on the 90th day the turtles carapace bones were restored with the formation of a full-fledged bone regenerate by the mechanism of indirect osteogenesis.

No evidence for a direct role of HLA-B27 in pathological bone formation in axial SpA

ObjectiveThe strong genetic association between HLA-B27 and ankylosing spondylitis has been known for over 40 years. HLA-B27 positivity is possibly associated with severity of ankylosis. We studied the in vitro...

Сurrent concepts in physiological and reparative osteogenesis

Studies conducted in the recent years by biologists strongly suggest that physiological and reparative osteogenesis, as well as of the functional, adaptive, and post-traumatic reconstruction of bone tissues are based on common and stereotypical molecular and cellular mechanisms. Our experimental studies have shown that all stages of the bone microstructure morphogenesis are synchronously and continuously associated with focal and stereotypical angiogenesis (capillarogenesis). A powerful factor in the implementation of reparative osteogenesis is the osteoinductive interaction of the ends of the damaged bone segments, which positively shows itself even in cases of large diastasis between the fragments (provided that the fragments are steadily fixed). After any kind of stable osteosynthesis, by ensuring the stability of the bone fragments for the entire period of consolidation, an endosteal cortical bone regeneration by direct osteogenesis (i.e. without fibro-cartilaginous tissue) is observed in the minimum amount at the shortest time period. Periosteal bone formation in this case is actually a reserve source of bone formation, which becomes effective during insufficiently stable conditions. The instability of the bone damage area particularly that of the metal implants results in the most severe destructive consequences.

Osteopontin-deficiency disturbs direct osteogenesis in the process of achieving osseointegration following immediate placement of endosseous implants.

The role of osteopontin (OPN) in the process of achieving osseointegration following implantation remains to be clarified. This study aimed to analyze the healing patterns of the bone-implant interface after immediate placement of implants in the maxillae of 4-week-old Opn-knockout (KO) and wild-type (WT) mice. After maxillary first molars were extracted, cavities were prepared with a drill and titanium implants blasted with ceramic abrasives containing hydroxyapatite/β-tricalcium phosphate were placed. Following fixation at 3, 5, 7, and 28 days after implantation, the samples were analyzed using immunohistochemistry, in situ hybridization, and an electron probe micro analyzer. Two types of bone healing were observed in the process of achieving osseointegration: "direct osteogenesis," where bone formation occurs at the implant surface, and "indirect osteogenesis," where it does at the pre-existing damaged bone surface in the WT mice. Direct osteogenesis occurred after the recruitment of tartrate resistant acid phosphatase-positive cells and the deposition of OPN on the implant surface. In contrast, the rate of osseointegration or direct osteogenesis was significantly low, and cell proliferation was disturbed in the Opn-KO mice. These results suggest that Opn-deficiency disturbs direct osteogenesis to lead the delayed osseointegration after immediate placement of endosseous implants.

Sustained dual release of placental growth factor-2 and bone morphogenic protein-2 from heparin-based nanocomplexes for direct osteogenesis.

ObjectiveTo compare the direct osteogenic effect between placental growth factor-2 (PlGF-2) and bone morphogenic protein-2 (BMP-2).MethodsThree groups of PlGF-2/BMP-2-loaded heparin–N-(2-hydroxyl) propyl-3-trimethyl ammonium chitosan chloride (HTCC) nanocomplexes were prepared: those with 0.5 μg PlGF-2; with 1.0 μg BMP-2; and with 0.5 μg PlGF-2 combined with 1.0 μg BMP-2. The loading efficiencies and release profiles of these growth factors (GFs) in this nanocomplex system were quantified using enzyme-linked immunosorbent assay, their biological activities were evaluated using cell counting kit-8, cell morphology, and cell number counting assays, and their osteogenic activities were quantified using alkaline phosphatase and Alizarin Red S staining assays.ResultsThe loading efficiencies were more than 99% for the nanocomplexes loaded with just PlGF-2 and for those loaded with both PlGF-2 and BMP-2. For the nanocomplex loaded with just BMP-2, the loading efficiency was more than 97%. About 83%–84% of PlGF-2 and 89%–91% of BMP-2 were stably retained on the nanocomplexes for at least 21 days. In in vitro biological assays, PlGF-2 exhibited osteogenic effects comparable to those of BMP-2 despite its dose in the experiments being lower than that of BMP-2. Moreover, the results implied that heparin-based nanocomplexes encapsulating two GFs have enhanced potential in the enhancement of osteoblast function.ConclusionPlGF-2-loaded heparin–HTCC nanocomplexes may constitute a promising system for bone regeneration. Moreover, the dual delivery of PlGF-2 and BMP-2 appears to have greater potential in bone tissue regeneration than the delivery of either GFs alone.

An Endochondral Ossification-Based Approach to Bone Repair: Chondrogenically Primed Mesenchymal Stem Cell-Laden Scaffolds Support Greater Repair of Critical-Sized Cranial Defects Than Osteogenically Stimulated Constructs In Vivo.

The lack of success associated with the use of bone grafts has motivated the development of tissue engineering approaches for bone defect repair. However, the traditional tissue engineering approach of direct osteogenesis, mimicking the process of intramembranous ossification (IMO), leads to poor vascularization. In this study, we speculate that mimicking an endochondral ossification (ECO) approach may offer a solution by harnessing the potential of hypertrophic chondrocytes to secrete angiogenic signals that support vasculogenesis and enhance bone repair. We hypothesized that stimulation of mesenchymal stem cell (MSC) chondrogenesis and subsequent hypertrophy within collagen-based scaffolds would lead to improved vascularization and bone formation when implanted within a critical-sized bone defect in vivo. To produce ECO-based constructs, two distinct scaffolds, collagen-hyaluronic acid (CHyA) and collagen-hydroxyapatite (CHA), with proven potential for cartilage and bone repair, respectively, were cultured with MSCs initially in the presence of chondrogenic factors and subsequently supplemented with hypertrophic factors. To produce IMO-based constructs, CHA scaffolds were cultured with MSCs in the presence of osteogenic factors. These constructs were subsequently implanted into 7 mm calvarial defects on Fischer male rats for up to 8 weeks in vivo. The results demonstrated that IMO- and ECO-based constructs were capable of supporting enhanced bone repair compared to empty defects. However, it was clear that the scaffolds, which were previously shown to support the greatest cartilage formation in vitro (CHyA), led to the highest new bone formation (p < 0.05) within critical-sized bone defects 8 weeks postimplantation. We speculate this to be associated with the secretion of angiogenic signals as demonstrated by the higher VEGF protein production in the ECO-based constructs before implantation leading to the greater blood vessel ingrowth. This study thus demonstrates the ability of recapitulating a developmental process of bone formation to develop tissue-engineered constructs that manifest appreciable promise for bone defect repair.

Use of Mesenchymal Stem Cells to Grow Cartilage and Bone

Regardless of the fact that bones have a tremendous capacity to be repaired, there are various clinical situations where allographs seek to enhance the regeneration of bones through promotion of osteogenesis [1]. Precarious botches linked to the present grafting treatments include osteonecrosis along with restricted combination between the grafted and host tissues. It is considered that the fundamental issue with the present approaches to bone grafting is the fact that they lead to bone regeneration by means of direct osteogenesis. Therefore, it may be hypothesized that the use of cartilage in the promotion of endochondral regeneration of bones may leverage normal development and repair sequences resulting in a properly vascularized regenerate capable of integrating with the host tissues. Various tests have demonstrated that cartilage grafts are able to support the regeneration of vascularized and integrated bone tissues in vivo, while lineage tracing experiments have revealed graft derived regenerates.

In vitro osteogenic potential of human mesenchymal stem cells is predicted by Runx2/Sox9 ratio.

Runx2 is one of the most studied transcription factors expressed in mesenchymal stem cells (MSCs) upon their commitment toward an osteogenic differentiation. During endochondral bone formation in vivo, Sox9 directly interacts with Runx2 and represses its activity; however, the role of Sox9 in direct osteogenesis in vitro has been largely overlooked. Bone marrow-derived human MSCs (hMSCs) were cultured in vitro either in the control or osteogenic medium supplemented with dexamethasone (DEX). To further investigate the role of Sox9 in direct osteogenesis in vitro, hMSCs were treated with Sox9 siRNA. We show here that Sox9 is the key early indicator during in vitro osteogenic differentiation of hMSCs. Osteogenic induction leads to a significant decrease of Sox9 gene and protein expression by day 7. Treatment of hMSCs with Sox9 siRNA enhanced mineralization in vitro, suggesting that downregulation of Sox9 is involved in direct osteogenesis. siRNA knockdown of Sox9 did not in itself induce osteogenesis in the absence of DEX, indicating that other factors are still required. Screening of not preselected donors of different ages and gender (n=12) has shown that the Runx2/Sox9 ratio on day 7 is correlated to the (45)Ca incorporation on day 28. The impact of Sox9 downregulation in the mineralization of human MSCs in vitro indicates a so far unprecedented role of Sox9 as a major regulator of direct osteogenesis. We propose that the Runx2/Sox9 ratio is a promising, early, in vitro screening method for osteogenicity of human MSCs.