RANKL-RANK signaling regulates osteoblast differentiation and bone formation

In the recent two decades, it has been well elucidated that receptor activator of nuclear factor-κB ligand （RANKL; also known as TNFSF11） binding to its receptor RANK （also known as TNFRSF11A） drives osteoclast development as the crucial signaling pathway.;However, accumulating evidence also implies that

miR-23a/b regulates the balance between osteoblast and adipocyte differentiation in bone marrow mesenchymal stem cells

Age-related osteoporosis is associated with the reduced capacity of bone marrow mesenchymal stem cells （BMSCs） to differentiate into osteoblasts instead of adipocytes. However, the molecular mechanisms that decide the fate of BMSCs remain unclear. In our study, microRNA-23a, and microRNA-23b （miR-23a/b） were found to be markedly downregulated in BMSCs of aged mice and humans. The overexpression of miR-23a/b in BMSCs promoted osteogenic differentiation, whereas the inhibition of miR-23a/b increased adipogenic differentiation. Transmembrane protein 64 （Tmem64）, which has expression levels inversely related to those of miR-23a/b in aged and young mice, was identified as a major target of miR-23a/b during BMSC differentiation. In conclusion, our study suggests that miR-23a/b has a critical role in the regulation of mesenchymal lineage differentiation through the suppression of Tmem64.

^(99m)TC-Methylene diphosphonate uptake at injury site correlates with osteoblast differentiation and mineralization during bone healing in mice

99mTc-Methylene diphosphonate （99mTc-MDP） is widely used in clinical settings to detect bone abnormalities. However, the mechanism of 99mTc-MDP uptake in bone is not well elucidated. In this study, we utilized a mouse tibia injury model, single-photon emission computed tomography （gamma scintigraphy or SPECT）, ex vivo micro-computed tomography, and histology to monitor 99mTc-MDP uptake in injury sites during skeletal healing. In an ex vivo culture system, calvarial cells were differentiated into osteoblasts with osteogenic medium, pulsed with 99mTc-MDP at different time points, and quantitated for 99mTc-MDP uptake with a gamma counter. We demonstrated that 99mTc-MDP uptake in the injury sites corresponded to osteoblast generation in those sites throughout the healing process. The 99mTc-MDP uptake within the injury sites peaked on day 7 post-injury, while the injury sites were occupied by mature osteoblasts also starting from day 7. ~mTc-MDP uptake started to decrease 14 days post-surgery, when we observed the highest level of bony tissue in the injury sites. We also found that 99mTc-MDP uptake was associated with osteoblast maturation and mineralization in vitro. This study provides direct and biological evidence for 99mTc-MDP uptake in osteoblasts during bone healing in vivo and in vitro.

3D printing of osteocytic Dll4 integrated with PCL for cell fate determination towards osteoblasts in vitro

Since 3D printed hard materials could match the shape of bone,cell survival and fate determination towards osteoblasts in such materials have become a popular research target.In this study,a scaffold of hardmaterial for 3D fabrication was designed to regulate developmental signal(Notch)transduction guiding osteoblast differentiation.We established a polycaprolactone(PCL)and cell-integrated 3D printing system(PCI3D)to reciprocally print the beams of PCL and cell-laden hydrogel for a module.This PCI3D module holds good cell viability of over 87%,whereas cells show about sixfold proliferation in a 7-day culture.The osteocytic MLO-Y4 was engineered to overexpress Notch ligand Dll4,making up 25%after mixing with 75%stromal cells in the PCI3D module.Osteocytic Dll4,unlike other delta-like family members such as Dll1 or Dll3,promotes osteoblast differentiation and themineralization of primary mouse and a cell line of bone marrow stromal cells when cultured in a PCI3D module for up to 28 days.Mechanistically,osteocytic Dll4 could not promote osteogenic differentiation of the primary bone marrow stromal cells(BMSCs)after conditional deletion of the Notch transcription factor RBPjκby Cre recombinase.These data indicate that osteocytic Dll4 activates RBPjκ-dependent canonical Notch signaling in BMSCs for their oriented differentiation towards osteoblasts.Additionally,osteocytic Dll4 holds a great potential for angiogenesis in human umbilical vein endothelial cells within modules.Our study reveals that osteocytic Dll4 could be the osteogenic niche determining cell fate towards osteoblasts.This will open a new avenue to overcome the current limitation of poor cell viability and low bioactivity of traditional orthopedic implants.

Aβ40 Promotes the Osteoblastic Differentiation of Aortic Valve Interstitial Cells through the RAGE Pathway

Amyloid beta(AB)peptide 40 enhances the activation of receptor for advanced glycation end products(RAGE)in immune-inflammatory diseases.RAGE exhibits several ffects in the setting of numerous cardiovascular events.We bypothesized that the Aβ40/RAGE pathway is involved in the osteoblastic differentiation of the valvular interstitial cell(VIC)phenotype,and RAGE knockout intervention could reduce the calcification of aortic valve interstitial cells(AVICs)by inhibiting the extracellular-regulated kinase1/2(ERK 1/2)/nuclear factor kappa-B(NF-kB)signaling pathway.To test this hypothesis,the activation of AB40/RAGE pathway in human calcific AVs was evaluated with immunohistochemical staining.Cultured calcific VIC models were used in vitro.The VICs were stimulated using Aβ40,with or without RAGE small interfering ribonucleic acid(siRNA),and ERK1/2 and NF-κB inhibitors for analysis.Our data revealed that AB40 induced the ERK 1/2/NF-κB signaling pathway and osteoblastic differentiation of AVICs via the RAGE pathway in vitro.

Wnt7b can replace Ihh to induce hypertrophic cartilage vascularization but not osteoblast differentiation during endochondral bone development

Indian hedgehog （Ihh） is an essential signal that regulates endochondral bone development. We have previously shown that Wnt7b promotes osteoblast differentiation during mouse embryogenesis, and that its expression in the perichondrium is dependent on Ihh signaling. To test the hypothesis that Wnt7b may mediate some aspects of Ihh function during endochondral bone development, we activated Wnt7b expression from the R26-Wnt7b allele with Col2-Cre in the Ihh-/- mouse. Artificial expression of Wnt7b rescued vascularization of the hypertrophic cartilage in the Ihh-/- mouse, but failed to restore orthotopic osteoblast differentiation in the perichondrium. Similarly, Wnt7b did not recover Ihh-dependent perichondral bone formation in the Ihh-/-; Gli3-/- embryo. Interestingly, Wnt7b induced bone formation at the diaphyseal region of long bones in the absence of Ihh, possibly due to increased vascularization in the area. Thus, Ihh-dependent expression of Wnt7b in the perichondrium may contribute to vascularization of the hypertrophic cartilage during endochondral bone development.

Polydopamine Coating on Titanium Affects Osteoblastic Differentiation to a Greater Degree than Does Surface Roughness

Biointerface design can greatly influence cell behavior. Therefore, in this study we examined the effects of three surface characteristics, roughness, chemistry, and wettability, on osteoblastic cell differentiation. We examined osteoblastic differentiation on titanium (Ti) samples with four levels of roughness (average roughness: 148.6 ± 23.1, 42 ± 6.2, 14.3 ± 5.5, 7.2 ± 1.6 nm) with or without a nanolayer coating of polydopamine (PDA). In vitro osteogenic differentiation was evaluated by quantifying alkaline phosphatase (AP) activity of human fetal preosteoblastic (hFOB 1.19) cells. The change in surface chemistry of Ti samples as a result of PDA coating was assessed by XPS analysis and water contact angle measurement. Results demonstrated that PDA treated samples were more hydrophilic, compared to untreated samples, and this was substrate roughness independent. Moreover, with the exception of the substrate with an oriented texture of surface nanotopography (RTi-4), the presence of a PDA nanolayer increased AP activity independent of substrate roughness. Our results suggest that surface chemistry and wettability, induced by a PDA nanolayer coating, had a greater effect on osteoblastic differentiation than did surface roughness.

Wnt signaling:Essential roles in osteoblast differentiation,bone metabolism and therapeutic implications for bone and skeletal disorders

Wnt signaling executes an indispensable performance in osteoblast differentiation,bone development,homeostasis,and remodeling.Wnt signals trigger the intracellular Wnt signaling cascade to initiate regulating the implication of b-catenin in the bone environment.Going through the novel discoveries done via high-throughput sequencing technologies on ge-netic mouse models,we highlighted the significant contribution of Wnt ligands,co-receptors,inhibitors,their related skeletal phenotypes in mouse models and the similar bone disorders clinically observed in human beings.Moreover,the crosstalk between Wnt signaling pathway and BMP,TGF-b,FGF,Hippo,Hedgehog,Notch and PDGF signaling pathways is thoroughly demonstrated to be the underlying gene regulatory network that orchestrates osteoblast dif-ferentiation and bone development.We also introspected the significance of Wnt signaling transduction in the reorganization of cellular metabolism by stimulating glycolysis,glutamine catabolism,and fatty acid oxidation in osteoblast-lineage cells that display an important reg-ulatory arbor in the cellular bioenergetics of the bone.Throughout this evaluation,most to date therapeutical approaches towards osteoporosis and other bone maladies found in human beings,are formulated with an aspiration to holistically revamp the present clinical applica-tions involving various monoclonal antibodies therapies that lack specificity,efficacy,and safety into more requisite advanced therapeutics that satisfy these three requirements for further clinical considerations.Conclusively,our review provides comprehensive scientific findings related to the fundamental significance of Wnt signaling cascades in skeletal system and the underlying gene regulatory network with other signaling pathways enlightening re-searchers with the possibility to further integrate the identified target molecules into thera-peutic strategies for skeletal disorders treatment in the clinic.

Effects of dexamethasone on proliferation, differentiation and apoptosis of adult human osteoblasts in vitro

Objective To observe the effects of dexamethasone on proliferation, differentiation and apoptosis of adult human osteoblasts in vitro.Methods Iliac trabecular bone specimens were obtained from adult patients undergoing necessary surgery. After the bone pieces were digested with collagenase-trypsin, osteoblasts were released and incubated at 37℃in a relative humidity of 95% and 5% CO2. Then, the cells were purified, and their passages were given DMEM-F12 and fetal bovine serum medium. Subsequently, 10^(-8) mol/L dexamethasone was added into the culture medium to incubate the osteoblasts for three days, and the cells from control groups were incubated without any drugs. All cells were observed continually with phase contrast microscope and transmission electron microscope. Finally, apoptosis was detected by the use of terminal deoxynucleotidyl transferase (TdT)-mediated dUTP nick end labeling (TUNEL) and biochemical indices, alkaline phosphatase (ALP) and osteocalcin (OCN) were used to determine the effects of dexamethasone on proliferation, differentiation and apoptosis of adult osteoblasts in vitro.Results In the adult osteoblasts obtained by collagenase-trypsin digestion, it achieved high survial, stable biochemical indices and excellent purification. Under the condition of dexamethasone 10^(-8) mol/L and osteoblasts 10 000/ml, there was significant promotion of ALP and OCN secretion without cell apoptosis.Conclusions Dexamethasone has a significant effect on the proliferation and differentiation of adult osteoblasts in vitro without apoptosis, and dexamethasone at the suggested concentration can be used as positive control in drug studies for osteoporosis treatment.

Aqueous extract of freeze-dried Protaetia brevitarsis larvae promotes osteogenesis by activating β-catenin signaling

Objective:To investigate the effect of an aqueous extract of Protaetia brevitarsis(AEPB)on osteogenesis using preosteoblast MC3T3-E1 cells and zebrafish larvae.Methods:Flow cytometric analysis was used to measure the cytotoxicy.Alkaline phosphatase activity was detetmined using p-nitrophenyl phosphate as a substrate.Calcium deposition was detected using alizarin red staining along with osteogenic marker expression in preosteoblast MC3T3E1 cells.In addition,vertebral formation in zebrafish larvae was detected using calcein staining and osteogenic gene expression.Results:AEPB highly promoted the expression of osteogenic markers including runt-related transcription factor 2,osterix,and alkaline phosphatase,along with elevated levels of mineralization in MC3T3-E1 cells.Moreover,AEPB accelerated vertebral formation in zebrafish larvae accompanied by upregulated expression of osteogenic genes.FH535,an inhibitor of Wnt/β-catenin,suppressed AEPB-induced osteogenic gene expression and vertebral formation,indicating that AEPB stimulates osteogenesis by activating the Wnt/β-catenin signaling pathway.Conclusions:AEPB stimulates osteoblast differentiation and bone formation by activatingβ-catenin.Therefore,AEPB is a promising material that induces osteogenesis,and is useful for the treatment of bone resorption diseases.

Rapidly deforming gastric carcinosarcoma with osteoblastic component:An autopsy case report

Carcinosarcomas are rare,malignant,biphasic tumors simultaneously comprising carcinoma and sarcoma in a single tumor.We present an extremely rare case of gastric carcinosarcoma with an osteoblastic component that drastically changed its shape within 2 mo.A 59-year-old male patient presented to the emergency outpatient unit with a complaint of black stool.Gastrointestinal endoscopy showed an ulcerated mass in the cardia of the lesser curvature of the stomach.Biopsy specimens revealed only adenocarcinoma.Two months later,the ulcerated lesion drastically changed its shape into an exophytic tumor.Total gastrectomy was performed.In the resected specimen,the gastric tumor contained both adenocarcinoma and sarcoma components with lace-like osteoid.The patient died 7 mo after the operation,and an autopsy was performed.In the autopsy,widespread metastases were present in the liver,lung,lymph nodes and peritoneum.In this report,we describe a case of gastric carcinosarcoma and presume its tumorigenesis based on the autopsy findings.

Combined effects of fluoride and arsenic exposure on proliferation,differentiation and beta-catenin expression in rat osteoblasts

<正>Objective To investigate the combined effects of fluoride(Na F)and arsenate(Na As O2)exposure on proliferation,differentiation and bata-catenin expression in SD rat osteoblasts.Methods Osteoblasts were isolated from calvarias of twelve SD rats born in 1~3 days and cul-

Calycosin-7-O-β-D-glucopyranoside stimulates osteoblast differentiation through regulating the BMP/WNT signaling pathways

The iso fl avone calycosin-7-O-β-D-glucopyranoside(CG) is a principal constituent of Astragalus membranaceus(AR) and has been reported to inhibit osteoclast development in vitro and bone loss in vivo. The aim of this study was to investigate the osteogenic effects of CG and its underlying mechanism in ST2 cells. The results show that exposure of cells to CG in osteogenic differentiation medium increases ALP activity, osteocalcin(Ocal) m RNA expression and the osteoblastic mineralization process. Mechanistically, CG treatment increased the expression of bone morphogenetic protein 2(BMP-2), p-Smad 1/5/8, β-catenin and Runx2, all of which are regulators of the BMP- or wingless-type MMTV integration site family(WNT)/β-catenin-signaling pathways. Moreover, the osteogenic effects of CG were inhibited by Noggin and DKK-1 which are classical inhibitors of the BMP and WNT/β-catenin-signaling pathways, respectively. Taken together, the results indicate that CG promotes the osteoblastic differentiation of ST2 cells through regulating the BMP/WNT signaling pathways. On this basis, CG may be a useful lead compound for improving the treatment of bone-decreasing diseases and enhancing bone regeneration.

PIP5k1β controls bone homeostasis through modulating both osteoclast and osteoblast differentiation

PIP5k1βis crucial to the generation of phosphotidylinosotol(4,5)P2.PIP5k1βparticipates in numerous cellular activities,such as B cell and platelet activation,cell phagocytosis and endocytosis,cell apoptosis,and cytoskeletal organization.In the present work,we aimed to examine the function of PIP5k1βin osteoclastogenesis and osteogenesis to provide promising strategies for osteoporosis prevention and treatment.We discovered that PIP5k1β deletion in mice resulted in obvious bone loss and that PIP5k1β was highly expressed during both osteoclast and osteoblast differentiation.Deletion of the gene was found to enhance the proliferation and migration of bone marrow-derived macrophage-like cells to promote osteoclast differentiation.PIP5k1β-/-osteoclasts exhibited normal cytoskeleton architecture but stronger resorption activity.PIP5kip deficiency also promoted activation of mitogen-activated kinase and Akt signaling,enhanced TRAF6 and c-Fos expression,facilitated the expression and nuclear translocation of NFATC1,and upregulated Grb2 expression,thereby accelerating osteoclast differentiation and function.Finally,PIP5k1β enhanced osteoblast differentiation by upregulating master gene expression through triggering smad1/5/8 signaling.Therefore,PIP5k1βmodulates bone homeostasis and remodeling.

miRNA expression profile during fluid shear stress-induced osteogenic differentiation in MC3T3-E1 cells

Background Mechanical stress plays an important role in the maintenance of bone homeostasis. Current hypotheses suggest that interstitial fluid flow is an important component of the system by which tissue level strains are amplified in bone. This study aimed to test the hypothesis that the short-term and appropriate fluid shear stress （FSS） is expected to promote the terminal differentiation of pre-osteoblasts and detect the expression profile of microRNAs in the FSS-induced osteogenic differentiation in MC3T3-E1 cells. Methods MC3T3-E1 cells were subjected to 1 hour of FSS at 12 dyn/cm2 using a parallel plate flow system. After FSS treatment, cytoskeleton immunohistochemical staining and microRNAs （miRNAs） were detected immediately. Osteogenic gene expression and immunohistochemical staining for collagen type I were tested at the 24th hour after treatment, alkaline phosphatase （ALP） activity assay was performed at 24th, 48th, and 72th hours after FSS treatment, and Alizarin Red Staining was checked at day 12. Results One hour of FSS at 12 dyn/cm2 induced actin stress fiber formation and rearrangement, up-regulated osteogenic gene expression, increased ALP activity, promoted synthesis and secretion of type I collagen, enhanced nodule formation, and promoted terminal differentiation in MC3T3-E1 cells. During osteogenic differentiation, expression levels of miR-20a, -21, -19b, -34a, -34c, -140, and -200b in FSS-induced cells were significantly down-regulated. Conclusion The short-term and appropriate FSS is sufficient to promote terminal differentiation of pre-osteoblasts and a group of miRNAs may be invovled in FSS-induced pre-osteoblast differentiation.

Induction of osteoblast differentiation in human adipose derived stem cells by lanthanum ions

Adipose derived stem cells represent a readily available source of adult stem cells for various biomedical applications. In this study, the proliferation and osteogenic differentiation potential of lanthanum nitrate（La3＋） on human adipose derived mesenchymal stem cells（hADSCs） were investigated for the first time and compared with that of dexamethasone（Dex）. Our results provided evidence that La3＋ at 50 μmol/L concentration promoted proliferation of hADSCs upto 2.4 fold when treated for 21 d in DMEM medium. Treatment of hADSCs with La3＋ containing osteogenic induction medium（α-MEM with ascorbic acid and β-glycerophosphate） for 7 d resulted in higher calcium deposition than that in the presence of Dex（0.1 μmol/L） as shown by Alizarin red S and von Kossa staining. Scanning electron micrographs also showed more extracellular matrix mineralization in the presence of La3＋. After 7 d of treatment with La3＋（10 μmol/L） the expression of RunX2, osteopontin（OP） and osteocalcin（OC） increased 3.4, 5.5 and 2.7 fold respectively. Our results provided evidence that in the presence of La3＋ osteogenic differentiation occurred earlier than that in the presence of Dex.

Directing the osteoblastic and chondrocytic differentiations of mesenchymal stem cells:matrix vs.induction media

While both induction culture media and matrix have been reported to regulate the stem cell fate,little is known about which factor plays a more decisive role in directing the MSC differentiation lineage as well as the underlying mechanisms.To this aim,we seeded MSCs on HA-collagen and HA-synthetic hydrogel matrixes,which had demonstrated highly different potentials toward osteoblastic and chondrocytic differentiation lineages,respectively,and cultured them with osteogenic,chondrogenic and normal culture media,respectively.A systematic comparison has been carried out on the effects of induction media and matrix on MSC adhesion,cytoskeleton organization,proliferation,and in particular differentiation into the osteoblastic and chondrocytic lineages.The results demonstrated that the matrix selection had a much more profound effect on directing the differentiation lineage than the induction media did.The strong modulation effect on the transcription activities might be the critical factor contributing to the above observations in our study,where canonical Wnt-b-Catenin signal pathway was directly involved in the matrix-driven osteoblastic differentiation.Such findings not only provide a critical insight on natural cellular events leading to the osteoblastic and chondrocytic differentiations,but also have important implications in biomaterial design for tissue engineering applications.

Control of MSC Differentiation by Tuning the Alkyl Chain Length of Phe- nylboroinc Acid Based Low-molecular-weight Gelators

The physical environment plays a critical role in modulating stem cell differentiation into specific lineages. In this study, we designed and synthesized a series of low-molecular-weight gels （LMWGs） with different moduli based on phenylboronic acid derivatives. The moduli of the LMWGs were readily tuned by varying the alkyl chain without any chemical crosslinker. The cell responses to the gels were evaluated with mesenchymal stem cell （MSCs）, in respect of cell morphology, proliferation and differentiation. The prepared gels were non-toxic to MSCs, suggesting good biocompatibility. The hydrogel stiffness exerted a striking modulation effect on MSC fate decisions, where MSCs were inclined to differentiate into osteoblasts in stiff LMWGs and into chondrocytes in soft LMWGs. The pivotal elastic modulus of the LMWGs to drive MSC differentiation into osteoblastic lineage and chondrocytic lineage were approximately 20 kPa - 40 kPa and 1 kPa - 10 kPa, respectively. Overall, our results demonstrated that the modification ofhydrogel stiffness via tuning the alkyl chain was a simple but effective approach to regulate MSC differentiation into specific lineage, which might have important implications in the design of LMWGs for tissue engineering applications.

Bioadaptive Nanorod Topography of Titanium Surface to Control Cell Behaviors and Osteogenic Differentiation of Preosteoblast Cells

Titanium （Ti） nanorods fabricated using selective corrosion of Ti substrate by anodic technology show better biocompatibility with pre-osteoblast cells. The current study investigated the response of the murine pre-osteoblast cell MCST3-E1 on Ti nanorod topography and untreated Ti surfaces by means of examination of the morphology and osteogenic differentiation responsible for the pre-osteoblast reaction. The morphology of MCST3-E1 cells was observed using scanning electron microscopy, and alkaline phosphatase （ALP） activity was measured using a colorimetric assay after incubation for 7, 14, and 21 days. The expression of three osteogenic differentiation markers including ALP, osteocalcin （OCN）, and collagen type 1A1 （COL1A1） and two transcription factors including runt related transcription factor 2 （Runx2） and osterix （Osx） at different time points was detected using real-time polymerase chain reaction analysis in both groups. Osx was used to confirm the protein level. The results showed that Ti nanorod surfaces provided prolonged higher levels of ALP activity compared with unmodified Ti surface on the 14th and 21st days. Gene expression analysis of ALP, OCN, and COL1A1 showed significant upregulation with modified nanorod topography after incubation for 14 and 21 days. Osteogenic transcription factors of Runx2 and Osx exhibited changes consistent with the osteogenic differentiation markers, and this may contribute to the persistently active differentiation of MC3T3-E1 cells in the Ti nanorod group. These results demonstrated that the current nanostructured surface may be considered bioadaptive topography to control cellular behaviors and osteoblast differentiation. The in vivo performance and applicability are further required to investigate osseointegration between implant and host bone in the early stages for prevention of aseptic implant loosening.

A new osteogenic protein isolated from Dioscorea opposita Thunb accelerates bone defect healing through the mTOR signaling axis

Delayed bone defect repairs lead to severe health and socioeconomic impacts on patients. Hence, there are increasing demands for medical interventions to promote bone defect healing. Recombinant proteins such as BMP-2 have been recognized as one of the powerful osteogenic substances that promote mesenchymal stem cells (MSCs) to osteoblast differentiation and are widely applied clinically for bone defect repairs. However, recent reports show that BMP-2 treatment has been associated with clinical adverse side effects such as ectopic bone formation, osteolysis and stimulation of inflammation. Here, we have identified one new osteogenic protein, named ‘HKUOT-S2’ protein, from Dioscorea opposita Thunb. Using the bone defect model, we have shown that the HKUOT-S2 protein can accelerate bone defect repair by activating the mTOR signaling axis of MSCs-derived osteoblasts and increasing osteoblastic biomineralization. The HKUOT-S2 protein can also modulate the transcriptomic changes of macrophages, stem cells, and osteoblasts, thereby enhancing the crosstalk between the polarized macrophages and MSCs-osteoblast differentiation to facilitate osteogenesis. Furthermore, this protein had no toxic effects in vivo. We have also identified HKUOT-S2 peptide sequence TKSSLPGQTK as a functional osteogenic unit that can promote osteoblast differentiation in vitro. The HKUOT-S2 protein with robust osteogenic activity could be a potential alternative osteoanabolic agent for promoting osteogenesis and bone defect repairs. We believe that the HKUOT-S2 protein may potentially be applied clinically as a new class of osteogenic agent for bone defect healing.