The alteration of a mechanical property of bone cells during the process of changing from osteoblasts to osteocytes

Osteocytes acquire their stellate shape during the process of changing from osteoblasts in bone. Throughout this process, dynamic cytoskeletal changes occur. In general, changes of the cytoskeleton affect cellular mechanical properties. Mechanical properties of living cells are connected with their biological functions and physiological processes. In this study, we for the first time analyzed elastic modulus, a mechanical property of bone cells. Bone cells in embryonic chick calvariae and in isolated culture were identified using fluorescently labeled phalloidin and OB7.3, a chick osteocyte-specific monoclonal antibody, and then observed by confocal laser scanning microscopy. The elastic modulus of living cells was analyzed with atomic force microscopy. To examine the consequences of focal adhesion formation on the elastic modulus, cells were pretreated with GRGDS and GRGES, and then the elastic modulus of the cells was analyzed. Focal adhesions in the cells were visualized by immunofluorescence of vinculin. From fluorescence images, we could distinguish osteoblasts, osteoid osteocytes and mature osteocytes both in vivo and in vitro. The elastic modulus of peripheral regions of cells in all three populations was significantly higher than in their nuclear regions. The elastic modulus of the peripheral region of osteoblasts was 12053+/-934 Pa, that of osteoid osteocytes was 7971+/-422 Pa and that of mature osteocytes was 4471+/-198 Pa. These results suggest that the level of elastic modulus of bone cells was proportional to the stage of changing from osteoblasts to osteocytes. The focal adhesion area of osteoblasts was significantly higher than that of osteocytes. The focal adhesion area of osteoblasts was decreased after treatment with GRGDS, however, that of osteocytes was not. The elastic modulus of osteoblasts and osteoid osteocytes were decreased after treatment with GRGDS. However, that of mature osteocytes was not changed. There were dynamic changes in the mechanical property of elastic modulus and in focal adhesions of bone cells.     

The effect of in vivo mechanical loading on estrogen receptor alpha expression in rat ulnar osteocytes.

The presence of estrogen receptor alpha (ER alpha) in osteocytes was identified immunocytochemically in transverse sections from 560 to 860 microm distal to the midshaft of normal neonatal and adult male and female rat ulnas (n = 3 of each) and from adult male rat ulnas that had been exposed to 10 days of in vivo daily 10-minute periods of cyclic loading producing peak strains of either -3000 (n = 3) or -4000 microstrain (n = 5). Each animal ambulated normally between loading periods, and its contralateral ulna was used as a control. In animals in which limbs were subject to normal locomotor loading alone, 14 +/- 1.2% SEM of all osteocytes in each bone section were ER alpha positive. There was no influence of either gender (p = 0.725) or age (p = 0.577) and no interaction between them (p = 0.658). In bones in which normal locomotion was supplemented by short periods of artificial loading, fewer osteocytes expressed ER alpha (7.5 +/- 0.91% SEM) than in contralateral control limbs, which received locomotor loading alone (14 +/- 1.68% SEM; p = 0.01; median difference, 6.43; 95% CI, 2.60, 10.25). The distribution of osteocytes expressing ER alpha was uniform across all sections and thus did not reflect local peak strain magnitude. This suggests that osteocytes respond to strain as a population, rather than as individual strain-responsive cells. These data are consistent with the hypothesis that ER alpha is involved in bone cells' responses to mechanical strain. High strains appear to decrease ER alpha expression. In osteoporotic bone, the high strains assumed to accompany postmenopausal bone loss may reduce ER alpha levels and therefore impair the capacity for appropriate adaptive remodeling.     

内质网应激与骨细胞及相关骨病的研究进展

内质网（endoplasmic reticulum, ER)作为细胞内重要的膜性结构,负责分泌蛋白与膜蛋白的折叠加工,脂类的生物合成以及钙平衡的调节。当ER环境发生改变,如缺血、缺氧、突变蛋白的大量堆积时,内质网应激（endoplasmic reticulum stress, ERS)启动,并引发未折叠蛋白反应、内质网超负荷反应和固醇调节级联反应。在应激状态下,ER对维持细胞内稳态起到至关重要的作用。一方面ERS有助于成骨细胞、破骨细胞和软骨细胞的生长,可以促进骨髓间充质干细胞向成骨细胞的分化,并抑制细胞凋亡的发生。另一方面在高强度长时间应激状态下,ERS无法维持细胞稳态,会通过多种信号通路诱导细胞凋亡的发生,加速细胞更新。根据近几年已有报道的文献研究整理,在骨质疏松、成骨不全、氟骨症等相关骨病的研究中,ERS同样发挥着重要的调节作用。特别是在发病早期,ERS通过对多种骨细胞的调节,缓解病情。当病情严重ERS会触发内质网凋亡信号,导致细胞凋亡和生物体的损伤。本文就近年来国内外对ERS与骨细胞及相关骨病的研究进展进行综述。     

PTH receptor signaling in osteocytes governs periosteal bone formation and intracortical remodeling

The periosteal and endocortical surfaces of cortical bone dictate the geometry and overall mechanical properties of bone. Yet the cellular and molecular mechanisms that regulate activity on these surfaces are far from being understood. Parathyroid hormone (PTH) has profound effects in cortical bone, stimulating periosteal expansion and at the same time accelerating intracortical bone remodeling. We report herein that transgenic mice expressing a constitutive active PTH receptor in osteocytes (DMP1‐caPTHR1 mice) exhibit increased cortical bone area and an elevated rate of periosteal and endocortical bone formation. In addition, DMP1‐caPTHR1 mice display a marked increase in intracortical remodeling and cortical porosity. Crossing DMP1‐caPTHR1 mice with mice lacking the Wnt coreceptor, LDL‐related receptor 5 (LRP5), or with mice overexpressing the Wnt antagonist Sost in osteocytes (DMP1‐Sost mice) reduced or abolished, respectively, the increased cortical bone area, periosteal bone formation rate, and expression of osteoblast markers and Wnt target genes exhibited by the DMP1‐caPTHR1 mice. In addition, DMP1‐caPTHR1 lacking LRP5 or double transgenic DMP1‐caPTHR1;DMP1‐Sost mice exhibit exacerbated intracortical remodeling and increased osteoclast numbers, and markedly decreased expression of the RANK decoy receptor osteoprotegerin. Thus, whereas Sost downregulation and the consequent Wnt activation is required for the stimulatory effect of PTH receptor signaling on periosteal bone formation, the Wnt‐independent increase in osteoclastogenesis induced by PTH receptor activation in osteocytes overrides the effect on Sost. These findings demonstrate that PTH receptor signaling influences cortical bone through actions on osteocytes and defines the role of Wnt signaling in PTH receptor action. © 2011 American Society for Bone and Mineral Research.     

Ubiquitin ligase Cbl-b downregulates bone formation through suppression of IGF-I signaling in osteoblasts during denervation.

Unloading can prevent bone formation by osteoblasts. To study this mechanism, we focused on a ubiquitin ligase, Cbl-b, which was highly expressed in osteoblastic cells during denervation. Our results suggest that Cbl-b may mediate denervation-induced osteopenia by inhibiting IGF-I signaling in osteoblasts. INTRODUCTION: Unloading, such as denervation (sciatic neurectomy) and spaceflight, suppresses bone formation by osteoblasts, leading to osteopenia. The resistance of osteoblasts to growth factors contributes to such unloading-mediated osteopenia. However, a detailed mechanism of this resistance is unknown. We first found that a RING-type ubiquitin ligase, Cbl-b, was highly expressed in osteoblastic cells after sciatic neurectomy in mice. In this study, we reasoned that Cbl-b played an important role in the resistance of osteoblasts to IGF-I. Materials AND METHODS: Cbl-b-deficient (Cbl-b(-/-)) or wildtype (Cbl-b(+/+)) mice were subjected to sciatic neurectomy. Bone formation in these mice was assessed by calcein labeling and histomorphometric analyses. We examined IGF-I signaling molecules in femora of these mice by Western blot and immunohistochemical analyses. We also examined the mitogenic response of Cbl-b-overexpressing or -deficient osteoblastic cells to various growth factors. RESULTS: In Cbl-b(+/+) mice, denervation decreased femur mass and bone formation, whereas it increased the expression of Cbl-b protein in osteoprogenitor cells and in osteocalcin-positive cells (osteoblastic cells) in hindlimb bone. In contrast, in Cbl-b(-/-) mice, bone mass and bone formation were sustained during denervation. Denervation inhibited the mitogenic response of osteoprogenitor cells most significantly to IGF-I. Therefore, we focused on Cbl-b-mediated modification of IGF-I signaling. Denervation decreased the amounts of insulin receptor substrate-1 (IRS-1), phosphatidly inositol 3-phosphate kinase (PI3K), and Akt-1 proteins in femora of Cbl-b(+/+) mice, whereas the amounts of these IGF-I signaling molecules in femora of Cbl-b(-/-) mice were constant after denervation. On a cellular level, primary osteoblastic cells from Cbl-b(-/-) mice were more stimulated to proliferate by IGF-I treatment compared with those from Cbl-b(+/+) mice. Furthermore, overexpression of Cbl-b increased ubiquitination and degradation of IRS-1 in primary Cbl-b(-/-) osteoblastic cells, leading to their impaired mitogenic response to IGF-I. CONCLUSIONS: These results suggest that Cbl-b induces resistance of osteoblasts to IGF-I during denervation by increasing IRS-1 degradation and that Cbl-b-mediated modification of IGF-I signaling may contribute to decreased bone formation during denervation.     

Bone morphogenetic proteins in bone stimulate osteoclasts and osteoblasts during bone development.

In this study, overexpression of noggin, a BMP antagonist, in developing bone caused significantly decreased osteoclast number as well as bone formation rate, resulting in increased bone mass with immature bone quality. BMP signaling plays important roles in normal bone development and regulation of bone resorption. INTRODUCTION: Bone morphogenetic proteins (BMPs) act on various types of cells. Although involvement of BMP signals in osteoblast differentiation has been studied extensively, the effects of BMPs on osteoclasts have not been widely researched. Consequently, the net effects of BMPs on bone remain unclear. The purpose of this study was to delineate more fully the role of BMPs in skeletal biology. MATERIALS AND METHODS: We generated transgenic mice that express BMP4 or noggin in bone under the control of the 2.3-kb alpha1(I) collagen chain gene (Col1a1) promoter, and analyzed their bone phenotype. We also analyzed bone of transgenic mice expressing BMP4 specifically in cartilage. RESULTS: Mice overexpressing BMP4 in bone developed severe osteopenia with increased osteoclast number. Mice overexpressing noggin, a BMP antagonist, in bone showed increased bone volume associated with decreased bone formation rate and decreased osteoclast number. The noggin-transgenic tibias exhibited reduced periosteal bone formation and reduced resorption of immature bone in marrow spaces, associated with frequent fractures at the diaphysis. Co-culture of primary osteoblasts prepared from noggin-transgenic calvariae and wildtype spleen cells resulted in poor osteoclast formation, which was rescued by addition of recombinant BMP2, suggesting that noggin inhibits osteoclast formation by attenuating BMP activities in noggin-transgenic mice. The expression levels of Rankl were not decreased in primary osteoblasts from noggin transgenic mice. Immunoblot analysis showed increased phosphorylation of Smad1/5/8 in osteoclast precursor cells after 20-minute treatment with BMPs, suggesting that these cells are stimulated by BMPs. Mice overexpressing BMP4 in cartilage had enlarged bones containing thick trabeculae, possibly because of expansion of cartilage anlagen. CONCLUSIONS: Overexpression of noggin in bone revealed that BMP signals regulate bone development through stimulation of osteoblasts and osteoclasts.     

Prevention of glucocorticoid induced-apoptosis of osteoblasts and osteocytes by protecting against endoplasmic reticulum (ER) stress in vitro and in vivo in female mice

Endoplasmic reticulum (ER) stress is associated with increased reactive oxygen species (ROS), results from accumulation of misfolded/unfolded proteins, and can trigger apoptosis. ER stress is alleviated by phosphorylation of eukaryotic translation initiation factor 2α (eIF2α), which inhibits protein translation allowing the ER to recover, thus promoting cell viability. We investigated whether osteoblastic cell apoptosis induced by glucocorticoids (GCs) is due to induction of ROS/ER stress and whether inhibition of eIF2α dephosphorylation promotes survival opposing the deleterious effects of GC in vitro and in vivo. Apoptosis of osteocytic MLO-Y4 and osteoblastic OB-6 cells induced by dexamethasone was abolished by ROS inhibitors. Like GC, the ER stress inducing agents brefeldin A and tunicamycin induced osteoblastic cell apoptosis. Salubrinal or guanabenz, specific inhibitors of eIF2α dephosphorylation, blocked apoptosis induced by either GC or ER stress inducers. Moreover, GC markedly decreased mineralization in OB-6 cells or primary osteoblasts; and salubrinal or guanabenz increased mineralization and prevented the inhibitory effect of GC. Furthermore, salubrinal (1 mg/kg/day) abolished osteoblast and osteocyte apoptosis in cancellous and cortical bone and partially prevented the loss of BMD at all sites and the decreased vertebral cancellous bone formation induced by treatment with prednisolone for 28 days (1.4 mg/kg/day). We conclude that part of the pro-apoptotic actions of GC on osteoblastic cells is mediated through ER stress, and that inhibition of eIF2α dephosphorylation protects from GC-induced apoptosis of osteoblasts and osteocytes in vitro and in vivo and from the deleterious effects of GC on the skeleton.     

Differentiation of cartilage from calvarial bone under the influence of bone matrix gelatin in vitro.

Fetal rat calvarium in an optimum (CMRL-1066) culture medium (with respect to amino acids. Tween 80, DNA precursors and hydrogen acceptors used in oxidative metabolism) produces new lamellar bone. The explanted osteoprogenitor cells survive, proliferate on living membrane bone surfaces and remodel membrane bone into lamellar bone. In the same culture medium, calvarial connective tissue and osteoprogenitor cells grow out of the membrane bone interstices onto a substratum consisting of bone matrix gelatin (BMG) and differentiate not into bone but into hyaline cartilage. Outgrowths from either bone or muscle onto a substratum of BMG prepared from bone autodigested in neutral buffer solutions, produce only fibrous connective tissue. In BGJ, a culture medium containing suboptimal ingredients for cell proliferation, calvarial bone produces neither lamellar bone nor new cartilage but is gradually resorbed and replaced by fibroblasts. In CMRL culture medium, outgrowths of mesenchymal-like cells from muscle onto a substratum of living membrane bone produce an epiphyseal plate-like columnar deposit of new hyaline cartilage. These observations suggest that the function of BMG is to evoke mesenchymal cell differentiation into prechondroblasts during the latent or migratory morphogenetic phase while the effect of the culture medium is to provide the bionutritional requirements for synthesis of hyaline cartilage matrix by chondrocytes during the patent phase of development.     

In situ imaging of the autonomous intracellular Ca(2+) oscillations of osteoblasts and osteocytes in bone

Bone cells form a complex three-dimensional network consisting of osteoblasts and osteocytes embedded in a mineralized extracellular matrix. Ca(2+) acts as a ubiquitous secondary messenger in various physiological cellular processes and transduces numerous signals to the cell interior and between cells. However, the intracellular Ca(2+) dynamics of bone cells have not been evaluated in living bone. In the present study, we developed a novel ex-vivo live Ca(2+) imaging system that allows the dynamic intracellular Ca(2+) concentration ([Ca(2+)](i)) responses of intact chick calvaria explants to be observed without damaging the bone network. Our live imaging analysis revealed for the first time that both osteoblasts and osteocytes display repetitive and autonomic [Ca(2+)](i) oscillations ex vivo. Thapsigargin, an inhibitor of the endoplasmic reticulum that induces the emptying of intracellular Ca(2+) stores, abolished these [Ca(2+)](i) responses in both osteoblasts and osteocytes, indicating that Ca(2+) release from intracellular stores plays a key role in the [Ca(2+)](i) oscillations of these bone cells in intact bone explants. Another possible [Ca(2+)](i) transient system to be considered is gap junctional communication through which Ca(2+) and other messenger molecules move, at least in part, across cell-cell junctions; therefore, we also investigated the role of gap junctions in the maintenance of the autonomic [Ca(2+)](i) oscillations observed in the intact bone. Treatment with three distinct gap junction inhibitors, 18α-glycyrrhetinic acid, oleamide, and octanol, significantly reduced the proportion of responsive osteocytes, indicating that gap junctions are important for the maintenance of [Ca(2+)](i) oscillations in osteocytes, but less in osteoblasts. Taken together, we found that the bone cells in intact bone explants showed autonomous [Ca(2+)](i) oscillations that required the release of intracellular Ca(2+) stores. In addition, osteocytes specifically modulated these oscillations via cell-cell communication through gap junctions, which maintains the observed [Ca(2+)](i) oscillations of bone cells.     

Prevention of glucocorticoid-induced apoptosis in osteocytes and osteoblasts by calbindin-D28k.

This study show for the first time that calbindin-D28k can prevent glucocorticoid-induced bone cell death. The anti-apoptotic effect of calbindin-D28k involves inhibition of glucocorticoid induced caspase 3 activation as well as ERK activation. INTRODUCTION: Recent studies have indicated that deleterious effects of glucocorticoids on bone involve increased apoptosis of osteocytes and osteoblasts. Because the calcium-binding protein calbindin-D28k has been reported to be anti-apoptotic in different cell types and in response to a variety of insults, we investigated whether calbindin-D28k could protect against glucocorticoid-induced cell death in bone cells. MATERIALS AND METHODS: Apoptosis was induced by addition of dexamethasone (dex; 10-6 M) for 6 h to MLO-Y4 osteocytic cells as well as to osteoblastic cells. Apoptosis percentage was determined by examining the nuclear morphology of transfected cells. Caspase 3 activity was evaluated in bone cells and in vitro. SELDI mass spectrometry (MS) was used to examine calbindin-D28k-caspase 3 interaction. Phosphorylation of calbindin-D28k was examined by 32P incorporation as well as by MALDI-TOF MS. ERK activation was determined by Western blot. RESULTS: The pro-apoptotic effect of dex in MLO-Y4 cells was completely inhibited in cells transfected with calbindin-D28k cDNA (5.6% apoptosis in calbindin-D28k transfected cells compared with 16.2% apoptosis in vector-transfected cells, p < 0.05). Similar results were observed in osteoblastic cells. We found that dex-induced apoptosis in bone cells was accompanied by an increase in caspase 3 activity. This increase in caspase 3 activity was inhibited in the presence of calbindin-D28k. In vitro assays indicated a concentration-dependent inhibition of caspase 3 by calbindin-D28k (Ki = 0.22 microM). Calbindin-D28k was found to inhibit caspase 3 specifically because the activity of other caspases was unaffected by calbindin-D28k. The anti-apoptotic effect of calbindin-D28k in response to dex was also reproducibly associated with an increase in the phosphorylation of ERK 1 and 2, suggesting that calbindin-D28k affects more than one signal in the glucocorticoid-induced apoptotic pathway. CONCLUSION: Calbindin-D28k, a natural non-oncogenic protein, could be an important target in the therapeutic intervention of glucocorticoid-induced osteoporosis.     

Low-intensity laser irradiation stimulates bone nodule formation via insulin-like growth factor-I expression in rat calvarial cells

BACKGROUND AND OBJECTIVE: We previously reported that low-intensity laser irradiation stimulated bone nodule formation through enhanced cellular proliferation and differentiation. However, the mechanisms of irradiation are unclear. Thus, we attempted to determine the responsibility of insulin-like growth factor (IGF)-I for the action observed. STUDY DESIGN/MATERIALS AND METHODS: Osteoblast-like cells were isolated from fetal rat calvariae and cultured with rat recombinant (r) IGF-I, IGF-I-antibody (Ab), and/or the cells were irradiated once (3.75 J/cm(2)) with a low-intensity Ga-Al-As laser (830 nm). The number and area of bone nodules formed in the culture were analyzed, and IGF-I expression was also examined. RESULTS: Treatment with rIGF-I significantly stimulated the number and area of bone nodules. This stimulatory effect was quite similar to those by laser irradiation, and this stimulation was abrogated dose-dependently by treatment with IGF-I-Ab. Moreover, laser irradiation significantly increased IGF-I protein and gene expression. CONCLUSION: The stimulatory effect of bone nodule formation by low-intensity laser irradiation will be at least partly mediated by IGF-I expression.     

β-半乳糖苷酶在体内外成骨细胞中的表达

目的 研究β—半乳糖苷酶(β—gal)在成骨细胞中的表达状况，为阐明MorquioB综合征的发病机制提供依据。方法 裸鼠各器官和骨组织标本行X-gal染色检测。抽取羊和人骨髓行骨髓基质细胞(BMSCs)培养，分为4组：I：Adv-hBMP-2转染组；Ⅱ：Adv—β—gal转染组；Ⅲ：未转染组；Ⅳ：地塞米松诱导组。分别行X-gal染色和RT-PCR检测β—gal的表达。结果 裸鼠骺板两侧、骨膜内面及松质骨的成骨细胞和破骨细胞可见多量β—gal的表达。未转染BMSCs组有少量β—gal的表达，其他3组细胞的β—gal表达增高。结论成骨细胞和破骨细胞可表达多量β—gal，该两种细胞的β—gal缺乏可能是MorquioB综合征骨骼异常的直接原因。     

Differentiation of osteoblasts and formation of mineralized bone in vitro

Summary Periostea consisting of the osteogenic layer and the fibrous layer of the periosteum were dissected from 17-day-old embryonic chick calvariae, leaving the osteoblasts behind on bone. The dissected periostea were folded with the osteogenic cells in apposition. The explants were cultured on plasma clots for up to 6 days, during which time osteodifferentiation was observed followed by osteoid formation in between the two layers. These cultures consistently mineralized in the presence of 5 or 10 mMβ-glycerophosphate. The mineralization and osteoid formation displayed many characteristics identical with those seen in vivo. Specifically, the osteoid formed was birefringent under polarized light, the central zone of osteoid became mineralized within 24 h of formation in vitro, and a clear border between mineralized and non-mineralized osteoid suggestive of a mineralization front was present. The unmineralized osteoid at the periphery was surrounded by osteoblasts. These data suggest that physiologic mineralization of osteoid produced in vitro did occur in this system by the addition of the alkaline phosphatase substrateβ-glycerophosphate.     

In vivo loading increases mechanical properties of scaffold by affecting bone formation and bone resorption rates

A successful bone tissue engineering strategy entails producing bone-scaffold constructs with adequate mechanical properties. Apart from the mechanical properties of the scaffold itself, the forming bone inside the scaffold also adds to the strength of the construct. In this study, we investigated the role of in vivo cyclic loading on mechanical properties of a bone scaffold. We implanted PLA/β-TCP scaffolds in the distal femur of six rats, applied external cyclic loading on the right leg, and kept the left leg as a control. We monitored bone formation at 7 time points over 35 weeks using time-lapsed micro-computed tomography (CT) imaging. The images were then used to construct micro-finite element models of bone-scaffold constructs, with which we estimated the stiffness for each sample at all time points. We found that loading increased the stiffness by 60% at 35 weeks. The increase of stiffness was correlated to an increase in bone volume fraction of 18% in the loaded scaffold compared to control scaffold. These changes in volume fraction and related stiffness in the bone scaffold are regulated by two independent processes, bone formation and bone resorption. Using time-lapsed micro-CT imaging and a newly-developed longitudinal image registration technique, we observed that mechanical stimulation increases the bone formation rate during 4-10 weeks, and decreases the bone resorption rate during 9-18 weeks post-operatively. For the first time, we report that in vivo cyclic loading increases mechanical properties of the scaffold by increasing the bone formation rate and decreasing the bone resorption rate.     

PRP modulates expression of bone matrix proteins in vivo without long-term effects on bone formation

This experimental study (domestic pig) examined the bone formation after filling defined defects of the frontal skull with autogenous bone or a deproteinized bovine bone matrix (DBBM) in combination with platelet-rich plasma (PRP). Six groups, both materials with and without PRP in two different concentrations (4.1x and 6.5x referring to untreated whole blood) were evaluated at 2, 4, 12, and 26 weeks by means of immunohistochemical staining for different bone matrix proteins, microradiography, light microscopy and polychromatic fluorescence labeling. The sequential expression of bone matrix proteins reflected the specific roles these proteins fulfil in the mineralization of hard tissue. Collagen I expression at 2 weeks was enhanced in all autogenous bone groups. No specific modification of the collagen I expression was found after use of DBBM with or without PRP. Osteopontin and especially osteonectin showed a remarkable enhancement at 4 weeks in nearly all autogenous bone and DBBM groups. These increased levels closely resembled the mineralization content evaluated by microradiography at that time. For the three autogenous bone groups, an expression peak for osteocalcin was demonstrated at 12 weeks, further reflecting the way of de novo bone formation. The microradiographic evaluation demonstrated a statistically significant enhancement in bone regeneration by PRP only after use of autogenous bone plus PRP at 2 weeks (P = 0.002). After 4 weeks, mineralization values after use of autogenous bone were significantly lower if PRP was added to the autogenous bone (P = 0.002). No long-term effects of the PRP administration were found in the mineralization process. In all DBBM groups, bone formation remained unchanged, confirming the lack of any osteoinductive capacity of PRP. PRP modulated the expression of bone matrix proteins in this experimental setting. However, an enhancement of bone formation was demonstrated only at 2 weeks after application of the higher PRP concentration in combination with autogenous bone. In conjunction with an anorganic bovine bone no effects of PRP on defect mineralization were discovered, demonstrating the lack of osteoinductive capacity in PRP as well as in DBBM.