Induction of chondrogenesis with a RANKL-binding peptide, WP9QY, in vitro and in vivo in a rabbit model.

WP9QY (W9) is a receptor activator of nuclear factor-κB ligand (RANKL)-binding peptide that inhibits osteoclastogenesis by blunting the RANKL-RANK interaction, and also increases osteoblastogenesis via RANKL reverse signaling. W9 has dual effects on osteoclasts and osteoblasts; however, it is unknown whether the peptide has an effect on chondrocytes. Here, we report that W9 induces proliferation and differentiation of chondrocytes in vitro and repairs full-thickness articular cartilage defects in vivo. W9 stimulated chondrocyte differentiation in a two-dimensional (2D) culture of human mesenchymal stem cells (hMSCs), and transforming growth factor ß3 (TGF-ß3) showed synergistic effects with W9 on chondrogenesis. W9 enlarged the size of 3D pellet cultures of hMSCs and produced chondrocyte-specific matrices, especially in combined treatment with TGF-ß3. The peptide also stimulated proliferation of hMSCs with induction of expression of chondrogenesis-related genes. Several RANKL inhibitors had no effect on chondrocytic differentiation. RANKL-knockdown experiments showed that W9 did not induce chondrogenesis through RANKL, but did induce osteoblastogenesis through RANKL. Intraarticular injection of W9 resulted in significant repair of full-thickness articular cartilage defects in rabbits. Taken together, these results suggest that W9 ameliorates the articular cartilage defects by increasing the volume of cartilaginous matrices with accompanying induction of proliferation and differentiation of chondrocytes via mechanisms independent of RANKL inhibition and RANKL reverse signaling. Since no pharmaceuticals are clinically available for treatment of cartilage damage such as osteoarthritis, our findings demonstrate the potential of W9 to address the unmet medical needs.

Significantly earlier ambulation and reduced risk of near-falls with continuous infusion nerve blocks: a retrospective pilot study of adductor canal block compared to femoral nerve block in total knee arthroplasty.

BACKGROUND: Near-falls should be detected to prevent falls related to the earlier ambulation after Total knee arthroplasty (TKA). The quadriceps weakness with femoral nerve block (FNB) has led to a focus on adductor canal block (ACB). We purposed to examine the risk of falls and the earlier ambulation in each continuous infusion nerve block. METHODS: Continuous infusion nerve block (FNB or ACB) was performed until postoperative day (POD) 2 or 3. Pain levels and falls/near-falls with knee-buckling were monitored from POD 1 to POD 3. The score on the manual muscle test, MMT (0 to 5, 5 being normal), of the patients who could ambulate on POD 1, was investigated. RESULTS: A total of 73 TKA cases, 36 FNB and 37 ACB, met the inclusion criteria. No falls were noted. But episodes of near-falls with knee-buckling were witnessed in 14 (39%) cases in the FNB group and in 4 (11%) in the ACB group (p = 0.0068). In the ACB group, 81.1% of patients could ambulate with parallel bars on POD 1, while only 44.4% of FNB patients could do so (p = 0.0019). The quadriceps MMT values in the ACB group was 2.82, significantly higher than 1.97 in the FNB group (p = 0.0035). There were no significant differences in pain as measured with a numerical rating scale (NRS) and rescue analgesia through POD 3. CONCLUSION: ACB was associated with significantly less knee-buckling and earlier ambulation post-TKA, with better quadriceps strength. Our study indicated the incidence of falls and near-falls with continuous infusion nerve blocks, and support the use of ACB to reduce the risk of falls after TKA. It is suggested that a certain number of the patients even with continuous ACB infusion should be considered with the effect of motor branch to prevent falls.

Irx3 and Bmp2 regulate mouse mesenchymal cell chondrogenic differentiation in both a Sox9-dependent and -independent manner.

Sox9, a master regulator of cartilage development, controls the cell fate decision to differentiate from mesenchymal to chondrogenic cells. In addition, Sox9 regulates the proliferation and differentiation of chondrocytes, as well as the production of cartilage-specific proteoglycans. The existence of Sox9-independent mechanisms in cartilage development remains to be determined. Here, we attempted to identify genes involved in such putative mechanisms via microarray analysis using a mouse chondrogenic cell line, N1511. We first focused on transcription factors that exhibited upregulated expression following Bmp2 treatment, which was not altered by subsequent treatment with Sox9 siRNA. Among these, we selected positive regulators for chondrogenesis and identified Iroquois-related homeobox 3 (Irx3) as one of the candidate genes. Irx3 expression gradually increased with chondrocyte terminal differentiation in a reciprocal manner to Sox9 expression, and promoted the chondrogenic differentiation of mesenchymal cells upon Bmp2 treatment. Furthermore, Irx3 partially rescued impaired chondrogenesis by upregulating the expression of epiphycan and lumican under reduced Sox9 expression. Finally, Irx3 was shown to act in concert with Bmp2 signaling to activate the p38 MAPK pathway, which in turn stimulated Sox9 expression, as well as the expression of epiphycan and lumican in a Sox9-independent manner. These results indicate that Irx3 represents a novel chondrogenic factor of mesenchymal cells, acts synergistically with Bmp2-mediated signaling, and regulates chondrogenesis independent of the transcriptional machinery associated with Sox9-mediated regulation.

Effect of epigallocatechin-3-gallate on the increase in type II collagen accumulation in cartilage-like MSC sheets.

With the aim to increase type II collagen content in the scaffold-free cartilage-like cell sheet using human bone marrow mesenchymal stem cells, we examined the effect of epigallocatechin-3-gallate (EGCG) addition to the chondrogenic medium for the cell sheet culture. The addition of EGCG (10 µM) increased the content of type II collagen 2-fold, while the addition did not markedly change the expression level of the genes encoding type II collagen and Sox 9. The reactive oxygen species level in the cells in cell sheets was thought to be too low to suppress the accumulation of type II collagen. On the other hand, the addition of EGCG markedly decreased both the matrix metalloproteinase-13 concentration in the supernatant of cell sheet culture and the type II collagen degradation activity in that supernatant. Taken together, EGCG may enhance the accumulation of type II collagen by suppressing type II collagen degradation.

Effect of epigallocatechin-3-o-gallate and quercetin on the cryopreservation of cartilage tissue.

The effects of epigallocatechin-3-o-gallate (EGCG) and quercetin on the contents of extracellular matrix (ECM) in porcine cartilage at 4 °C were investigated. The addition of quercetin at 0.01 mM for the incubation of porcine cartilage disks at 4 °C for 2 week could suppress the decrease in ECM and the compliance of the disks, markedly greater than those of EGCG (1.0 mM).

Disparate response of articular- and auricular-derived chondrocytes to oxygen tension.

PURPOSE/AIM: To determine the effect of reduced (5%) oxygen tension on chondrogenesis of auricular-derived chondrocytes. Currently, many cell and tissue culture experiments are performed at 20% oxygen with 5% carbon dioxide. Few cells in the body are subjected to this supra-physiological oxygen tension. Chondrocytes and their mesenchymal progenitors are widely reported to have greater chondrogenic expression when cultured at low, more physiological, oxygen tension (1-7%). Although generally accepted, there is still some controversy, and different culture methods, species, and outcome metrics cloud the field. These results are, however, articular chondrocyte biased and have not been reported for auricular-derived chondrocytes. MATERIALS AND METHODS: Auricular and articular chondrocytes were isolated from skeletally mature New Zealand White rabbits, expanded in culture and differentiated in high density cultures with serum-free chondrogenic media. Cartilage tissue derived from aggregate cultures or from the tissue engineered sheets were assessed for biomechanical, glycosaminoglycan, collagen, collagen cross-links, and lysyl oxidase activity and expression. RESULTS: Our studies show increased proliferation rates for both auricular and articular chondrocytes at low (5%) O2 versus standard (20%) O2. In our scaffold-free chondrogenic cultures, low O2 was found to increase articular chondrocyte accumulation of glycosaminoglycan, but not cross-linked type II collagen, or total collagen. Conversely, auricular chondrocytes accumulated less glycosaminoglycan, cross-linked type II collagen and total collagen under low oxygen tension. CONCLUSIONS: This study highlights the dramatic difference in response to low O2 of chondrocytes isolated from different anatomical sites. Low O2 is beneficial for articular-derived chondrogenesis but detrimental for auricular-derived chondrogenesis.

Usefulness of urinary CTX-II and NTX-I in evaluating radiological knee osteoarthritis : the Matsudai knee osteoarthritis survey.

BACKGROUND: To assess the usefulness of the urinary crosslinked C-telopeptide of type II collagen (uCTX-II) or crosslinked N-telopeptide of type I collagen (uNTX-I) for evaluating radiological knee osteoarthritis (OA), a cross-sectional study was conducted in the cohorts of the Matsudai knee osteoarthritis survey performed in Niigata, Japan. METHODS: Urine specimens and standing knee AP X-rays were obtained from 1040 subjects who provided informed consent. The relationship between these markers and gender, age (patients aged 40-59 or 60-79 years), use of bisphosphonates, and OA grades (K-L classification) were analyzed. The diagnostic ability of uCTX-II to detect radiological knee OA was confirmed in the over 60-year-old subjects using a ROC curve. RESULTS: The over 60-year-old men with OA grade 3,4 group had significantly higher uCTX-II levels than the other OA grade groups. In the over 60-year-old women, the uCTX-II levels significantly increased according to the progression of the knee OA grade. No significant difference was observed between the uNTX-I levels in the different OA grade groups. From the standpoint of biomarkers, the higher quartiles of the uCTX-II and uNTX-I levels gradually included higher numbers of grade ≥2 OA subjects in the over 60 year-old women. The area under the curve (AUC) in ROC analysis of uCTX-II exhibited a significant association with the diagnosis of knee OA in women (AUC 0.63), although the accuracy was evaluated to be low in the single measurement of our health checkup-based analysis. CONCLUSIONS: This population-based study indicates that the uCTX-II level is strongly correlated with the knee OA grade in women over age 60. A further analysis is needed to clarify its predictive accuracy.

[Cartilage repair and regenerative medicine ; past, present, and future].

Since the days of ancient Greece, it has been known that articular cartilage has poor potential to repair. In April, 2013, autologous chondrocyte implantation (ACI) was approved by the Japanese ministry of Health, Labour and Welfare, as the first orthopedic cell therapy in Japan. Half a century has passed since Chesterman's report on rabbit allogeneic chondrocyte implantation, and approximately 20 years since the first human ACI report by Brittberg. In Japan, 24 years has passed since Wakitani's allogeneic chondrocyte implantation, and over 10 years since the human ACI report by Ochi. A long-term follow-up clinical trial with the high statistical power is needed to verify the safety and efficacy of new cartilage joint therapy. Moreover, we need the infrastructure, including drug and/or device regulation and timely approved by the government, to apply new therapies in similar time frames to other developed nations.

Review of Caplan (1991) on cell-based therapeutic technology using Mesenchymal Stem Cells.

This classic discusses the original 1991 publication 'Mesenchymal Stem Cells (MSCs)' by Dr. Caplan on the emergence of a new therapeutic technology of self-cell repair using MSCs. After the original classic publication, a large number of methods to regenerate injured tissue have been reported. Currently, MSCs are used clinically to repair articular cartilage defects, liver cirrhosis, cerebral infarction, spinal cord injury, graft-versus-host disease and others. As a result, MSCs are considered one of the most important cell sources for regenerative medicine. An MSC has been demonstrated to be a multipotent stem cell in cell culture and was thought to contribute to the regeneration of injured tissue at transplant sites, but recently, the concept of MSCs has changed such that they are now referred to as "medicinal signaling cells," owing to their often indirect effects on tissue repair and regeneration. Regardless of the name, either mesenchymal stem cells or medicinal signaling cells, MSCs will be used to regenerate injured tissue more widely in the near future.

KUS121 attenuates the progression of monosodium iodoacetate-induced osteoarthritis in rats.

Currently there is no effective treatment available for osteoarthritis (OA). We have recently developed Kyoto University Substances (KUSs), ATPase inhibitors specific for valosin-containing protein (VCP), as a novel class of medicine for cellular protection. KUSs suppressed intracellular ATP depletion, endoplasmic reticulum (ER) stress, and cell death. In this study, we investigated the effects of KUS121 on chondrocyte cell death. In cultured chondrocytes differentiated from ATDC5 cells, KUS121 suppressed the decline in ATP levels and apoptotic cell death under stress conditions induced by TNFα. KUS121 ameliorated TNFα-induced reduction of gene expression in chondrocytes, such as Sox9 and Col2α. KUS121 also suppressed ER stress and cell death in chondrocytes under tunicamycin load. Furthermore, intraperitoneal administration of KUS121 in vivo suppressed chondrocyte loss and proteoglycan reduction in knee joints of a monosodium iodoacetate-induced OA rat model. Moreover, intra-articular administration of KUS121 more prominently reduced the apoptosis of the affected chondrocytes. These results demonstrate that KUS121 protects chondrocytes from stress-induced cell death in vitro and in vivo, and indicate that KUS121 is a promising novel therapeutic agent to prevent the progression of OA.

Endoglin is involved in BMP-2-induced osteogenic differentiation of periodontal ligament cells through a pathway independent of Smad-1/5/8 phosphorylation.

The periodontal ligament (PDL), a connective tissue located between the cementum of teeth and the alveolar bone of mandibula, plays a crucial role in the maintenance and regeneration of periodontal tissues. The PDL contains fibroblastic cells of a heterogeneous cell population, from which we have established several cell lines previously. To analyze characteristics unique for PDL at a molecular level, we performed cDNA microarray analysis of the PDL cells versus MC3T3-E1 osteoblastic cells. The analysis followed by validation by reverse transcription-polymerase chain reaction and immunochemical staining revealed that endoglin, which had been shown to associate with transforming growth factor (TGF)-beta and bone morphogenetic proteins (BMPs) as signaling modulators, was abundantly expressed in PDL cells but absent in osteoblastic cells. The knockdown of endoglin greatly suppressed the BMP-2-induced osteoblastic differentiation of PDL cells and subsequent mineralization. Interestingly, the endoglin knockdown did not alter the level of Smad-1/5/8 phosphorylation induced by BMP-2, while it suppressed the BMP-2-induced expression of Id1, a representative BMP-responsive gene. Therefore, it is conceivable that endoglin regulates the expression of BMP-2-responsive genes in PDL cells at some site downstream of Smad-1/5/8 phosphorylation. Alternatively, we found that Smad-2 as well as Smad-1/5/8 was phosphorylated by BMP-2 in the PDL cells, and that the BMP-2-induced Smad-2 phosphorylation was suppressed by the endoglin knockdown. These results, taken together, raise a possibility that PDL cells respond to BMP-2 via a unique signaling pathway dependent on endoglin, which is involved in the osteoblastic differentiation and mineralization of the cells.

Chondromodulin-1 directly suppresses growth of human cancer cells.

BACKGROUND: Chondromodulin-1 (ChM1), an endogenous anti-angiogenic factor expressed in cartilage, has been suggested to inhibit invasion of endothelial cells into cartilage. In addition, the ectopic administration of ChM1 has been reported to suppress tumorigenesis in vivo. However, it is unclear whether the anti-tumor effect is due to not only the anti-vascularization effect of ChM1, but also its direct action against oncocytes. In the present study, we sought to determine whether ChM1 has a direct action on tumor cells. METHODS: BrdU incorporation assay was performed on human umbilical vein endothelial cells (HUVECs), normal human dermal fibroblasts (NHDFs), HepG2 cells and HeLa cells in the presence or absence of recombinant human ChM1 (rhChM1). An adenovirus that expresses ChM1, Ad-ChM1, was established and applied to the tumor xenografted in vivo, and to in vitro tumor cells cultured on plates or in soft agar. Cell cycle-related proteins and the phosphorylation of Erk, Akt, and GSK3beta, the downstream molecules of the extracellular matrix-integrin signaling pathways, in HepG2 cells treated with or without Ad-ChM1 were detected by western blot analysis. Luciferase reporter assays of STAT, GAS, and ISRE, which participate in another cytokine signaling pathway, ware performed in HepG2, HeLa, and HUVEC cells. RESULTS: ChM1 suppressed BrdU incorporation in HUVECs and in HepG2 cells dose-dependently, but did not suppress BrdU incorporation in NHDFs and HeLa cells cultured on plates. In soft agar, however, ChM1 suppressed the growth of HeLa cells, as well as HepG2 cells. Western blot analyses demonstrated that ChM1 decreased the levels of cyclin D1, cyclin D3, and cdk6 and increased those of p21cip1 without affecting the phosphorylation levels of Erk, Akt, and GSK3beta in HepG2 cells. The luciferase reporter assay demonstrated that ChM1 suppressed the transcriptional activities of STAT and GAS but not of ISRE. CONCLUSION: ChM1 directly suppressed the proliferation of tumor cells in an anchorage-independent manner. However, ChM1 did not alter the phosphorylation of downstream molecules, at which the signaling pathways through growth factor and cytokine receptors converge with the anchorage-dependent pathway. Our results show that ChM1 has a direct anti-tumor effect; moreover, this effect occurs by inhibiting the STAT signaling pathway.

Relationship between radiological knee osteoarthritis and biochemical markers of cartilage and bone degradation (urine CTX-II and NTX-I): the Matsudai Knee Osteoarthritis Survey.

Biochemical markers of cartilage and bone degradation are becoming increasingly important in the evaluation of knee osteoarthritis (OA). To clarify the correlation between radiological knee OA and urine CTX-II (C-terminal crosslinking telopeptide of collagen type II) or urine NTX-I (N-terminal crosslinking telopeptide of type I collagen), we conducted a cross-sectional study in the cohorts of the epidemiological knee survey at the Matsudai district in Niigata Prefecture, Japan. Urine specimens were collected from 296 subjects, and CTX-II and NTX-I were measured using ELISA. Standing knee AP X-rays were obtained and graded according to the Kellgren-Lawrence classification. The subjects were then divided by gender, age (40- to 59-year-old group and 60- to 79-year-old group), and the X-ray grade (Grade 0, 1, Grade 2, and Grade 3, 4). In non-OA (Grade 0, 1) subjects, the 60- to 79-year-old group had significantly higher CTX-II values than the younger group only in females. The subjects of both genders aged over 60 years of age with OA Grade 3, 4 had significantly higher CTX-II values than the Grade 0, 1 group or the Grade 2 group. For NTX-I, there were no significant differences between each OA grade although the Grade 3, 4 group females from 60 to 79 years of age had higher values than the Grade 2 group. In addition, in the 60- to 79-year-old subjects of both genders, a positive correlation was observed between the urine CTX-II and urine NTX-I. For the subjects ranging from 60 to 79 years of age in both genders, the urine CTX-II values indicate the progression of OA. In addition, the weak but positive correlation between urine CTX-II and urine NTX-I in the subjects ranging from 60 to 79 years of age in both genders suggests that bone resorption and cartilage degradation appear to develop in parallel.

Transplantation of autologous bone marrow-derived mesenchymal stem cells under arthroscopic surgery with microfracture versus microfracture alone for articular cartilage lesions in the knee: A multicenter prospective randomized control clinical trial.

INTRODUCTION: To investigate the efficacy of the transplantation of autologous bone marrow-derived mesenchymal stem cells (BMSCs) under arthroscopy with microfracture (MFX) compared with microfracture alone. METHODS: Eleven patients with a symptomatic articular cartilage defect of the knee were included in the study. They were randomized to receive BMSCs with MFX (cell-T group, n=7) or MFX alone (control group, n=4). Clinical results were evaluated using International Knee Documentation committee (IKDC) knee evaluation questionnaires and the Knee Injury and Osteoarthritis Outcome Score (KOOS) before and 48 weeks after surgery. Quantitative and qualitative assessments of repair tissue were carried out at 48 weeks by T2 mapping of magnetic resonance images (MRIs) and the magnetic resonance observation of cartilage repair tissue (MOCART) scoring system with follow-up MRI. RESULTS: No significant differences between preoperative and postoperative IKDC and KOOS were observed in the cell-T or control group. However, forty-eight weeks after surgery, the cell-T group showed a trend for a greater KOOS QOL score compared with the control group (79.4 vs. 39.1, respectively; P=0.07). The T2 value did not differ significantly between the two groups, but the mean MOCART score was significantly higher in the cell-T group than in the control group (P=0.02). CONCLUSIONS: Compared with MFX alone, BMSC transplantation with MFX resulted in better postoperative healing of the cartilage and subchondral bone as determined by the MOCART score. Clinically, BMSC transplantation with MFX gave a higher KOOS QOL score after 48 weeks.

Effects of agitation rate on aggregation during beads-to-beads subcultivation of microcarrier culture of human mesenchymal stem cells.

With the aim to utilize human mesenchymal stem cells (hMSCs) grown in large scale for regenerative medicine, effects of agitation rate on aggregation during beads-to-beads subcultivation of microcarrier culture of hMSCs were studied. hMSCs could attach and grew on surface-type microcarriers of Cytodex 1, whereas almost no cell elongation and growth were observed on porous type microcarriers of Cytopores. The percentages of aggregated Cytodex 1 microcarriers at an agitation rate of 60 and 90 rpm were lower than that at 30 rpm, which was the lowest agitation rate necessary for the suspension of Cytodex 1 microcarriers, and the cells grew fastest at 60 rpm. hMSC could be subcultivated on Cytodex 1 by the beads-to-beads method at both 30 and 60 rpm without trypsinization. However, agitation at 60 rpm resulted in a markedly lower percentage of aggregated microcarriers not only before but also after subcultivation. The percentages of CD90- and CD166-positive cells among cells grown on Cytodex 1 at 60 rpm (91.5 and 87.6 %) were comparable to those of cells grown in the pre-culture on dishes. In conclusion, hMSCs could be subcultivated on Cytodex 1 by beads-to-beads method maintaining the expressions of the cell surface antigens CD90 and CD166, while adjusting agitation rate could decrease the microcarrier aggregation.

Quality Evaluation of Human Bone Marrow Mesenchymal Stem Cells for Cartilage Repair.

Quality evaluation of mesenchymal stem cells (MSCs) based on efficacy would be helpful for their clinical application. In this study, we aimed to find the factors of human bone marrow MSCs relating to cartilage repair. The expression profiles of humoral factors, messenger RNAs (mRNAs), and microRNAs (miRNAs) were analyzed in human bone marrow MSCs from five different donors. We investigated the correlations of these expression profiles with the capacity of the MSCs for proliferation, chondrogenic differentiation, and cartilage repair in vivo. The mRNA expression of MYBL1 was positively correlated with proliferation and cartilage differentiation. By contrast, the mRNA expression of RCAN2 and the protein expression of TIMP-1 and VEGF were negatively correlated with proliferation and cartilage differentiation. However, MSCs from all five donors had the capacity to promote cartilage repair in vivo regardless of their capacity for proliferation and cartilage differentiation. The mRNA expression of HLA-DRB1 was positively correlated with cartilage repair in vivo. Meanwhile, the mRNA expression of TMEM155 and expression of miR-486-3p, miR-148b, miR-93, and miR-320B were negatively correlated with cartilage repair. The expression analysis of these factors might help to predict the ability of bone marrow MSCs to promote cartilage repair.

Synergistic effect of ascorbic acid and collagen addition on the increase in type 2 collagen accumulation in cartilage-like MSC sheet.

Aiming to increase the content of type 2 collagen in scaffold-free cartilage-like cell sheets prepared using human bone marrow mesenchymal stem cells, the effect of several kinds of additives in a chondrogenic medium was investigated. Addition of ascorbic acid 2 phosphate (VCP) at a high concentration (250 µg/ml) and type 1 atelocollagen (5 µg/ml) increased the accumulation of type 2 collagen by fourfold and twofold, respectively. On the other hand, an antioxidant, glutathione showed no such effect. The synergistic effect of VCP and type 1 atelocollagen resulted in an eightfold increase in the accumulation level of type 2 collagen. Furthermore, the gene expression level of type 2 collagen increased and that of matrix metalloproteinase-13 (MMP-13) decreased to approximately one-third of the control. The increase in type 2 collagen accumulation in the scaffold-free cartilage-like cell sheet might be due to not only the enhancement of the synthesis but also the suppression of the degradation of type 2 collagen by MMP-13.

Osteoinduction with highly purified beta-tricalcium phosphate in dog dorsal muscles and the proliferation of osteoclasts before heterotopic bone formation.

The aim of the study was to examine the chronological histology of osteoinduction of highly purified beta-tricalcium phosphate (beta-TCP) implanted in dog dorsal muscles. Specimens were harvested on days 14, 28, 42, 56, 112 and 168 after implantation, and were analyzed by hematoxylin and eosin (HE) staining, tartrate-resistant acid phosphatase (TRAP) staining, immunohistochemistry, in situ hybridization, and silver impregnation. After day 28, abundant TRAP- and cathepsin K-positive multinucleated cells adhered to beta-TCP, suggesting that these cells are osteoclasts that can resorb beta-TCP. On day 56, new bone was formed and alpha1 chain of type I procollagen mRNA-positive osteoblasts lined the newly formed bone. Silver impregnation showed abundant collagen fibrils within the beta-TCP micropores. These results suggest that micropores function as a storage space for extracellular matrix components, including collagen. Newly formed bone never degenerated in the late stage, suggesting that beta-TCP has good biocompatibility and this material retains the conditions appropriate for osteointegration and bioresorption. In conclusion, beta-TCP has osteoinductivity after implantation in dog dorsal muscles without use of bone marrow cells or osteoinductive cytokines. The appearance of a large number of active osteoclasts precedes new bone formation.