Shock wave application enhances pertussis toxin protein-sensitive bone formation of segmental femoral defect in rats.

Extracorporeal shock waves (ESWs) elicit a dose-dependent effect on the healing of segmental femoral defects in rats. After ESW treatment, the segmental defect underwent progressive mesenchymal aggregation, endochondral ossification, and hard callus formation. Along with the intensive bone formation, there was a persistent increase in TGF-beta1 and BMP-2 expression. Pretreatment with pertussis toxin reduced ESW-promoted callus formation and gap healing, which presumably suggests that Gi proteins mediate osteogenic signaling. INTRODUCTION: Extracorporeal shock waves (ESWs) have previously been used to promote bone repair. In our previous report, we found that ESWs promoted osteogenic differentiation of mesenchymal cells through membrane perturbation and activation of Ras protein. In this report, we show that ESWs elicit a dose-dependent effect on the healing of segmental defects and that Gi proteins play an important role in mediating ESW stimulation. MATERIALS AND METHODS: Rats with segmental femoral defects were subjected to ESW treatment at different energy flux densities (EFD) and impulses. Bone mass (mineral density and calcium content), osteogenic activities (bone alkaline phosphatase activity and osteocalcin content), and immunohistochemistry were assessed. RESULTS: An optimal ESW energy (500 impulses at 0.16 mJ/mm2 EFD) stimulated complete bone healing without complications. ESW-augmented healing was characterized by significant increases (p < 0.01) in callus size, bone mineral density, and bone tissue formation. With exposure to ESW, alkaline phosphatase activity and osteocalcin production in calluses were found to be significantly enhanced (p < 0.05). After ESW treatment, the histological changes we noted included progressive mesenchymal aggregation, endochondral ossification, and hard callus formation. Intensive bone formation was associated with a persistent increase in transforming growth factor-beta 1 (TGF-beta1) and bone morphogenetic protein-2 (BMP-2) expression, suggesting both growth factors were active in ESW-promoted bone formation. We also found that pertussis toxin, an inhibitor of membrane-bound Gi proteins, significantly reduced (p < 0.01) ESW promotion of callus formation and fracture healing. CONCLUSION: ESW treatments enhanced bone formation and the healing of segmental femoral defects in rats. It also seems likely that TGF-beta1 and BMP-2 are important osteogenic factors for ESW promotion of fracture healing, presumably through Gi protein-mediated osteogenic signaling.     

Activation of extracellular signal-regulated kinase (ERK) and p38 kinase in shock wave-promoted bone formation of segmental defect in rats

Extracorporeal shock waves (ESW) have recently been used in bone repair. Extracellular signal-regulated kinase (ERK) and p38 kinase are found to act as important mediators for osteogenic factor and mechanical-stimulated proliferation and differentiation of bone-forming cells. A previous study reported that ESW promoted healing of segmental defects in rats by inducing bone morphogenetic proteins (Bone 32 (2003) 387-396) and stimulating osteogenic differentiation of mesenchymal stem cells. In this study, we found that ERK and p38 activation was involved in ESW-augmented bone regeneration of segmental defects. ESW treatment (0.16 mJ/mm2, 1 Hz, 500 impulses) rapidly promoted [3H]-thymidine uptake in 1 day and progressively increased alkaline phosphatase activity, collagen I, II, and osteocalcin synthesis in callus organ culture within 14 days after treatment. Results of [gamma-32P]-phosphotransferase activity assay showed that ERK and p38 in calluses were rapidly activated 1 day and 7 days after ESW treatment, respectively. Histological observation showed that segmental defects subjected to ESW treatment underwent typical bone formation (mesenchymal cell aggregation, hypertrophic cartilage, and endochondral/intramembrane ossification). Intensive bone formation coincided with evident expression of phosphorylated ERK and p38. Moreover, expression of phosphorylated ERK persisted in mesenchymal, chondral, and osteoblastic cells at newly developed bone and cartilage, and the expression of activated p38 was evident on chondral cells located at hypertrophic cartilage. Our findings suggest that mitogen-activated protein kinases (MAPK) regulate the stimulation of biophysical ESW, triggering mitogenic and osteogenic responses in the defects. ERK phosphorylation is active throughout the period of ESW-induced bone regeneration. p38 activation most likely plays an important role in signaling cartilage formation in callus.     

Distinct and Overlapping Patterns of Localization of Bone Morphogenetic Protein (BMP) Family Members and a BMP Type II Receptor During Fracture Healing in Rats

Bone morphogenetic proteins (BMPs) and their receptors (BMPRs) are thought to play an important role in bone morphogenesis. The purpose of this study was to determine the locations of BMP-2/-4, osteogenic protein-1 (OP-1, also termed BMP-7), and BMP type II receptor (BMPR-II) during rat fracture healing by immunostaining, and thereby elucidate the possible roles of the BMPs and BMPR-II in intramembranous ossification and endochondral ossification. In the early stage of fracture repair, the expression of BMP-2/-4 and OP-1 was strongly induced in the thickened periosteum near the fracture ends, and coincided with an enhanced expression of BMPR-II. On day 7 after fracture, staining for BMP-2/-4 and OP-1 immunostaining was increased in various types of chondrocytes, and was strong in fibroblast-like spindle cells and proliferating chondrocytes in endochondral bone. On day 14 after fracture, staining with OP-1 antibody disappeared in proliferating and mature chondrocytes, while BMP-2/-4 staining continued in various types of chondrocytes until the late stage. In the newly formed trabecular bone, BMP-2/-4 and OP-1 were present at various levels. BMPR-II was actively expressed in both intramembranous ossification and endochondral ossification. Additionally, immunostaining for BMP-2/-4 and OP-1 was observed in multinucleated osteoclast-like cells on the newly formed trabecular bone, along with BMPR-II. In reference to our previous study of BMP type I receptors (BMPR-IA and BMPR-IB), BMPR-II was found to be co-localized with BMPR-IA and BMPR-IB. BMP-2/-4 and OP-1 antibodies exhibited distinct and overlapping immunostaining patterns during fracture repair. OP-1 may act predominantly in the initial phase of endochondral ossification, while BMP-2/-4 acts throughout this process. Thus, these findings suggested that BMPs acting through their BMP receptors may play major roles in modulating the sequential events leading to bone formation.     

Recruitment of mesenchymal stem cells and expression of TGF-beta 1 and VEGF in the early stage of shock wave-promoted bone regeneration of segmental defect in rats.

Extracorporeal shock wave (ESW) treatment has recently been established as a method to enhance bone repair. Here, we reported that ESW-promoted healing of segmental defect via stimulation of mesenchymal stem cell recruitment and differentiation into bone forming cells. Rats with a segmental femoral defect were exposed to a single ESW treatment (0.16 mJ/mm(2), 1 Hz, 500 impulses). Cell morphology and histological changes in the defect region were assessed 3, 7, 14, and 28 days post-treatment. Presence of mesenchymal stem cell was assayed by immuno-staining for RP59, a recently discovered marker, and also production of TGF-beta 1 and VEGF was monitored. ESW treatment increased total cell density and the proportion of RP59 positive cells in the defect region. High numbers of round- and cuboidal-shaped cells strongly expressing RP59 were initially found. Later, the predominant cell type was spindle-shaped fibroblastic cells, subsequently, aggregates of osteogenic and chondrogenic cells were observed. Histological observation suggested that bone marrow stem cells were progressively differentiated into osteoblasts and chondrocytes. RP59 staining was initially intense and decreased with the appearance of expression depended on the differentiation states of osteogenic and chondrogenic cells during the regeneration phase. Mature chondrocytes and osteoblasts exhibited only slight RP59 immuno-reactivity. Expression of TGF-beta 1 and VEGF-A mRNA in the defect tissues was also significantly increased (P<0.05) after ESW treatment as determined by RT-PCR. Intensive TGF-beta 1 immuno-reactivity was induced immediately, whereas a lag period was observed for VEGF-A. Chondrocytes and osteoblasts at the junction of ossified cartilage clearly exhibited VEGF-A expression. Our findings suggest that recruitment of meseoblasts at the junction of ossified cartilage clearly exhibited mesenchymal stem cells is a critical step in bone reparation that is enhanced by ESW treatment. TGF-beta 1 and VEGF-A are proposed to play a chemotactic and mitogenic role in recruitment and differentiation of mesenchymal stem cells.     

Osteogenesis induced by extracorporeal shockwave in treatment of delayed osteotendinous junction healing

Healing at the osteotendinous junction (OTJ) is challenging in orthopedic surgery. The present study aimed to test extracorporeal shockwave (ESW) in treatment of a delayed OTJ healing. Twenty‐eight rabbits were used for establishing a delayed healing (DH) model at patella‐patellar‐tendon (PPT) complex after partial patellectomy for 4 weeks and then were divided into DH and ESW groups. In the ESW group, a single ESW treatment was given at postoperative week 6 to the PPT healing complex. The samples were harvested at week 8 and 12 for radiographic and histological evaluations with seven samples for each group at each time point. Micro‐CT results showed that new bone volume was 1.18 ± 0.61 mm³ in the ESW group with no measurable new bone in the DH group at postoperative week 8. Scar tissue formed at the OTJ healing interface of the DH group, whereas ESW triggered high expression of VEGF in hypertrophic chondrocytes at week 8 and regeneration of the fibrocartilage zone at week 12 postoperatively. The accelerated osteogenesis could be explained by acceleration of endochondral ossification. In conclusion, ESW was able to induce osteogenesis at OTJ with delayed healing with enhanced endochondral ossification process and regeneration of fibrocartilage zone. These findings formed a scientific basis to potential clinical application of ESW for treatment of delayed OTJ healing. © 2009 Orthopaedic Research Society. Published by Wiley Periodicals, Inc. J Orthop Res 28:70–76, 2010     

Expression of parathyroid hormone-related peptide and insulin-like growth factor I during rat fracture healing.

Parathyroid hormone-related peptide (PTHrP) and insulin-like growth factor I (IGF-I) are both involved in the regulation of bone and cartilage metabolisms and their interaction has been reported in osteoblasts. To investigate the interaction of PTHrP and IGF-I during fracture healing, the expression of mRNA for PTHrP and IGF-I, and receptors for PTH/PTHrP and IGF were examined during rat femoral fracture healing using an in situ hybridization method and an immunohistochemistry method, respectively. During intramembranous ossification, PTHrP mRNA, IGF-I mRNA and IGF receptors were detected in preosteoblasts, differentiated osteoblasts and osteocytes in the newly formed trabecular bone. PTH/PTHrP receptors were markedly detected in osteoblasts and osteocytes, but only barely so in preosteoblasts. During cartilaginous callus formation, PTHrP mRNA was expressed by mesenchymal cells and proliferating chondrocytes. PTH/PTHrP receptors were detected in proliferating chondrocytes and early hypertrophic chondrocytes. IGF-I mRNA and IGF receptor were co-expressed by mesenchymal cells, proliferating chondrocytes, and early hypertrophic chondrocytes. At the endochondral ossification front, osteoblasts were positive for PTHrP and IGF-I mRNA as well as their receptors. These results suggest that IGF-I is involved in cell proliferation or differentiation in mesenchymal cells, periosteal cells, osteoblasts and chondrocytes in an autocrine and/or paracrine fashion. Furthermore, PTHrP may be involved in primary callus formation presumably co-operating with IGF-I in osteoblasts and osteocytes, and by regulating chondrocyte differentiation in endochondral ossification.     

In vitro hypertrophy and calcification of human fracture haematoma-derived cells in chondrogenic differentiation

Purpose

The haematoma at a fracture site plays an important role in fracture healing. Previously, we demonstrated that a fracture haematoma contains multilineage mesenchymal progenitor cells. We postulated that the haematoma provided a source of chondrogenic cells for endochondral ossification during fracture healing and preservation of the cells contributed to biological fracture healing. In this study, we investigated whether haematoma-derived cells (HCs) could differentiate into hypertrophic chondrocytes and finally induce calcification of the extracellular matrix in vitro.

Methods

Fracture haematomas were obtained from four patients. HCs were cultured for five weeks under conditions that induce chondrogenic differentiation, followed by two weeks of hypertrophic induction using a pellet culture system. The pellets were analysed histologically and immunohistochemically. The gene expression levels of chondrogenic, hypertrophic, osteogenic, and angiogenic markers were measured by real-time PCR.

Results

The histological and immunohistochemical analyses revealed that HCs differentiated into chondrocytes and hypertrophic chondrocytes, followed by calcification of the extracellular matrix. This sequential differentiation was also reflected in the gene expression profiles. After chondrogenic induction, expression of osteogenic and angiogenic markers was not significantly upregulated. However, the expression of these markers was significantly upregulated following hypertrophic induction. These in vitro observations mimicked the process of endochondral ossification during fracture healing.

Conclusions

Our results suggest that the fracture haematoma may offer a source of cells with chondrogenic potential that play key roles in endochondral ossification during fracture healing. These findings support the opinion that the haematoma should be preserved for biological fracture healing.     

Expression of Indian Hedgehog During Fracture Healing in Adult Rat Femora

Indian hedgehog (Ihh) has recently been shown to be expressed in prehypertrophic and hypertrophic chondrocytes during embryonic development, and it has been implicated in the regulation of terminal differentiation of chondrocytes. In this paper we examined the expression of Ihh during fracture healing in an adult rat model. A transverse diaphyseal fracture was made in the right femur, and the expression of Ihh in the fracture callus was examined at 1, 2, and 3 weeks after fracture. Northern blot analysis demonstrated the expression of Ihh mRNA in these tissues. Immunohistological analysis detected hedgehog protein in prehypertrophic chondrocytes in the fracture callus at 1 week after fracture. From 2 weeks and on, positive staining was observed in hypertrophic chondrocytes as well. At 3 weeks, some of the osteoblasts close to the endochondral ossification front were also stained positive for hedgehog protein. Our data indicate that Ihh is expressed in chondrocytes and osteoblasts during the process of fracture healing in adult rat femora, suggesting that Ihh, a regulator of endochondral ossification in embryonic development, may also play a role in the regulation of bone formation during fracture repair in adult animals. Received: 29 March 1999 / Accepted: 30 September 1999     

Chondrocytes provide morphogenic signals that selectively induce osteogenic differentiation of mesenchymal stem cells.

During endochondral bone development cartilage formation always precedes that of bone, leading to the hypothesis that chondrocytes provide inductive signals for osteogenesis. To test this hypothesis, C3H10T1/2 mesenchymal stem cells were cocultured in membrane separated trans-well culture chambers with nonhypertrophic chondrocytes, hypertrophic chondrocytes, calvaria osteoblasts, or tendon fibroblasts derived from embryonic chickens to assess if individual cell types would selectively promote osteogenic differentiation. Then, differentiation of C3H10T1/2 mesenchymal stem cells in coculture were compared with that induced by bone morphogenetic protein 7 or osteogenic protein-1 (BMP-7; OP-1) treatment. Osteogenesis, as determined by the expression of Cbfa1 and osteocalcin (OC) messenger RNAs (mRNAs), was induced strongly in C3H10T1/2 cells cocultured with both chondrocyte cell populations but was not induced by coculture with either osteoblasts or skin fibroblasts. Interestingly, treatment of C3H10T1/2 cells with BMP-7 induced both chondrogenesis and osteogenesis, and only osteogenic differentiation was observed in the C3H10T1/2 cells cocultured with chondrocytes. No alterations in the expression of mRNAs for BMP-1 to -8 were observed in the C3H10T1/2 cells under any of the coculture conditions. This shows that the induction of endogenous BMPs by coculture does not regulate osteogenesis in an autocrine manner. These results show that chondrocytes express soluble morphogenetic factors that selectively promote osteogenesis, and this selective effect is not mimicked by an exogenously added BMP.     

Immunohistochemical localization of bone morphogenetic proteins (BMPs) 2, 4, 6, and 7 during induced heterotopic bone formation.

The distribution and staining intensity of bone morphogenetic proteins (BMPs) 2, 4, 6, and 7 were assessed by immunohistochemistry in ectopic bone induced in Nu/Nu mice by Saos-2 cell derived implants. Devitalized Saos-2 cells or their extracts can induce endochondral bone formation when implanted subcutaneously into Nu/Nu mice. BMP staining was mostly cytoplasmic. The most intense BMP staining was seen in hypertrophic and apoptotic chondrocytes, osteoprogenitor cells such as periosteal and perivascular cells, and osteoblasts. BMP staining in osteocytes and osteoclasts was variable, ranging from undetectable to intensely stained, and from minimal to moderately stained in megakaryocytes of the induced bone marrow. BMP-2, 4, 6, and 7 staining in Saos-2 implant-induced bone indicates the following: (1) Saos-2 cell products promote expression of BMPs by host osteoprogenitor cells, which in turn, leads to bone and marrow formation at ectopic sites; (2) strong BMP staining is seen in maturing chondrocytes, and thus may play a role in chondrocyte differentiation and/or apoptosis; (3) BMP expression in perivascular and periosteal cells indicates that osteoprogenitor cells also express BMP; (4) BMP release by osteoclasts may promote osteoblastic differentiation at sites of bone remodeling. These new data can be useful in understanding the role of BMPs in promoting clinical bone repair and in various pathologic conditions.     

Inhibition of growth plate angiogenesis and endochondral ossification with diminished expression of MMP-13 in hypertrophic chondrocytes in FGF-2-treated rats

Fibroblast growth factors (FGFs)/fibroblast growth factor receptor-3 signaling interferes with endochondral bone growth. However, the exact mechanisms by which FGFs inhibit endochondral ossification remain to be elucidated. In the present study, we utilized immunohistochemical techniques to clarify the effects of FGF-2 on the proximal tibial growth plate cartilage, when injected systemically into growing rats. In the FGF-2-treated rats, the growth plate was obviously thickened and, in the lowermost part, the hypertrophic chondrocytes were flattened, with an irregular arrangement. The connection of the cartilage columns and trabecular bone was disrupted. FGF-2 treatment stimulated the proliferation of chondrocytes and permitted their differentiation, but inhibited vascular invasion and resorption of the cartilage matrix. Expression of matrix metalloproteinase-13 (MMP-13) was detected in the chondrocytes in the last row of the hypertrophic zone of the growth plate in control animals. The immunoreactivity of MMP-13 was diminished in the regions where endochondral ossification was disturbed in the FGF-2-treated rats. Because MMP-13 has potent proteolytic activity on cartilage components, the FGF-2 signal may inhibit angiogenesis and endochondral ossification of the growth plate by the suppression of MMP-13 expression in hypertrophic chondrocytes. Received: March 17, 2001 / Accepted: November 16, 2001     

Localization of Smads, the TGF-beta family intracellular signaling components during endochondral ossification.

Members of the transforming growth factor-beta (TGF-beta) family transduce signals from the cell membrane to the nucleus via specific type I and type II receptors and Smad proteins. Smad1 and Smad5 mediate intracellular signaling of bone morphogenetic protein (BMP), whereas Smad2 and Smad3 transduce TGF-beta signaling. Smad4 is a common mediator required for both pathways. Smad6 and Smad7 inhibit signaling by members of the TGF-beta superfamily. Here, we examined the expression of Smad1 to Smad7 proteins during endochondral ossification of epiphyseal plate of growing rats using immunohistochemical techniques. The expression of Smad proteins was correlated with the expression of TGF-beta1 and its receptors, and BMP-2/4 and BMP receptors. The results show that TGF-beta1 and BMP-2/4 were actively expressed in chondrocytes that are undergoing proliferation and maturation, which overlaps with expression of their corresponding type I and type II receptors. The Smads, however, exhibited a distinct expression pattern, respectively. For example, Smad1 and Smad5 were highly expressed in proliferating chondrocytes and in those chondrocytes that are undergoing maturation. The TGF-beta/activin-restricted Smads were also expressed in a nearly complementary fashion; Smad2 was strongly expressed in proliferating chondrocytes, whereas Smad3 was strongly observed in maturing chondrocytes. Smad4 was broadly expressed in all zones of epiphyseal plate. Inhibitory Smads, Smad6 and Smad7, were strongly expressed in the zone of cartilage that contained mature chondrocytes. Our findings show a colocalization of the pathway-restricted and inhibitory Smads with activating ligands or ligands whose action they antagonize and their receptors in various zones of epiphyseal growth plate, suggesting that TGF-beta superfamily Smad signaling pathways plays a morphogenic role during endochondral bone formation.     

Acute phosphate restriction leads to impaired fracture healing and resistance to BMP‐2

Hypophosphatemia leads to rickets and osteomalacia, the latter of which results in decreased biomechanical integrity of bones, accompanied by poor fracture healing. Impaired phosphate‐dependent apoptosis of hypertrophic chondrocytes is the molecular basis for rickets. However, the underlying pathophysiology of impaired fracture healing has not been characterized previously. To address the role of phosphate in fracture repair, mice were placed on a phosphate‐restricted diet 2 days prior to or 3 days after induction of a mid‐diaphyseal femoral fracture to assess the effects of phosphate deficiency on the initial recruitment of mesenchymal stem cells and their subsequent differentiation. Histologic and micro‐computed tomographic (µCT) analyses demonstrated that both phosphate restriction models dramatically impaired fracture healing primarily owing to a defect in differentiation along the chondrogenic lineage. Based on Sox9 and Sox5 mRNA levels, neither the initial recruitment of cells to the callus nor their lineage commitment was effected by hypophosphatemia. However, differentiation of these cells was impaired in association with impaired bone morphogenetic protein (BMP) signaling. In vivo ectopic bone‐formation assays and in vitro investigations in ST2 stromal cells confirmed that phosphate restriction leads to BMP‐2 resistance. Marrow ablation studies demonstrate that hypophosphatemia has different effects on injury‐induced intramembranous bone formation compared with endochondral bone formation. Thus phosphate plays an important role in the skeleton that extends beyond mineralized matrix formation and growth plate maturation and is critical for endochondral bone repair. © 2010 American Society for Bone and Mineral Research     

Programmed removal of chondrocytes during endochondral fracture healing

This investigation tested the hypothesis that the removal of chondrocytes during endochondral fracture healing involves an ordered process of programmed cell death. To accomplish this, unilateral closed fractures were created in the femora of 36 Sprague-Dawley rats. The rats were killed in groups of four on days 1, 3, 7, 14, 21, 28, 42, 49, and 56 after fracture. The femora were embedded in paraffin and tested for expression of specific markers of fragmented DNA with use of a terminal deoxyuridyl transferase-mediated deoxyuridine triphosphatebiotin nick end labeling (TUNEL) technique. To determine the potential for trans–differentiation of chondrocytes to osteoblasts calluses were also hybridized to detect expression of osteocal in mRNA. Cell proliferation was assessed by an immunohistochemical detection method for proliferating cell nuclear antigen. A separate group of four rats was killed on day 28 to represent the later stage of the endochondral ossification, and the calluses were examined for cellular morphology with transmission electron microscopy. The results showed a coordination in both time and space of the activities of cellular proliferation and programmed cell death. Cell proliferation was most active in the earlier phases of fracture healing (days 1 through 14) although TUNEL expression was apparent in hypertrophic chondrocytes on day 14 after fracture and persisted until day 28. In the later stages of fracture healing (days 14 through 28), proliferating cell nuclear antigen was no longer synthesized in hard callus (intramembranous bone) and cell removal was the dominant activity in soft callus chondrocytes. Expression of osteocalcin mRNA was detected in osteoblasts but not in hypertrophic chondrocytes or in any other nonosteoblastic cell type. These findings support the hypothesis that the removal of chondrocytes during endochondral fracture healing is part of an ordered transition of tissue types in which the cellular mechanisms are genetically programmed to involve proliferation, maturation, and apoptotic cell death.     

Midkine is expressed during repair of bone fracture and promotes chondrogenesis.

Midkine (MK) is a heparin-binding growth/differentiation factor implicated in the control of development and repair of various tissues. Upon fracture of the murine tibia, MK was found to be transiently expressed during bone repair. MK was immunohistochemically detected in spindle-shaped mesenchymal cells at the fracture site on day 4 after fracture and in chondrocytes in the area of endochondral ossification on day 7. MK expression was decreased on day 14 and scarcely seen on day 28 when bone repair was completed. This mode of MK expression is reminiscent of MK expression during development. MK was expressed in hypertrophic chondrocytes of the prebone cartilage rudiments on embryonic day 14 in mouse embryos. MK was also strongly expressed in the epiphyseal growth plate. MK was localized intracellularly during both bone repair and development, and this localization was confirmed by immunoelectron microscopy for embryonic chondrocytes. When MK cDNA was transfected into ATDC5 chondrogenic cells and overexpressed, the majority of transfected cells with strong MK expression showed enhanced chondrogenesis as revealed by increased synthesis of sulfated glycosaminoglycans, aggrecan, and type II collagen. These results suggest that MK plays important roles in chondrogenesis and contributes to bone formation and repair.     

Prostaglandin E2 inhibits BMP signaling and delays chondrocyte maturation

While cyclooxygenases are important in endochondral bone formation during fracture healing, mechanisms involved in prostaglandin E2 (PGE2) regulation of chondrocyte maturation are incompletely understood. The present study was undertaken to determine if PGE2 effects on chondrocyte differentiation are related to modulation of the bone morphogenetic protein (BMP) signaling pathway. In primary murine sternal chondrocytes, PGE2 differentially regulated genes involved in differentiation. PGE2 induced type II collagen and MMP-13, had minimal effects on alkaline phosphatase, and inhibited the expression of the maturational marker, type X collagen. In BMP-2–treated cultures, PGE2 blocked the induction of type X collagen. All four EP receptors were expressed in chondrocytes and tended to be inhibited by BMP-2 treatment. RCJ3.1C5.18 chondrocytes transfected with the protein kinase A (PKA) responsive reporter, CRE-luciferase, showed luciferase induction following exposure to PGE2, consistent with activation of PKA signaling and the presence of the EP2 and EP4 receptors. Both PGE2 and the PKA agonist, dibutyryl cAMP, blocked the induction of the BMP-responsive reporter, 12XSBE, by BMP-2 in RCJ3.1C5.18 chondrocytes. In contrast, PGE2 increased the ability of TGF-β to activate the TGF-β-responsive reporter, 4XSBE. Finally, PGE2 down-regulated BMP-mediated phosphorylation of Smads 1, 5, and 8 in RCJ3.1C5.18 cells and in primary murine sternal chondrocytes. Altogether, the findings show that PGE2 regulates chondrocyte maturation in part by targeting BMP/Smad signaling and suggest an important role for PGE2 in endochondral bone formation. © 2008 Orthopaedic Research Society. Published by Wiley Periodicals, Inc. J Orthop Res 27: 785–792, 2009     

Amniotic membrane attenuates heterotopic ossification following high-dose bone morphogenetic protein-2 treatment of segmental bone defects

Treatment of large bone defects with supraphysiological doses of bone morphogenetic protein-2 (BMP-2) has been associated with complications including heterotopic ossification (HO), inflammation, and pain, presumably due to poor spatiotemporal control of BMP-2. We have previously recapitulated extensive HO in our rat femoral segmental defect model by treatment with high-dose BMP-2 (30 μg). Using this model and BMP-2 dose, our objective was to evaluate the utility of a clinically available human amniotic membrane (AM) around the defect space for guided bone regeneration and reduction of HO. We hypothesized that AM surrounding collagen sponge would attenuate heterotopic ossification compared with collagen sponge alone. In vitro, AM retained more BMP-2 than a synthetic poly(ε-caprolactone) membrane through 21 days. In vivo, as hypothesized, the collagen + AM resulted in significantly less heterotopic ossification and correspondingly, lower total bone volume (BV), compared with collagen sponge alone. Although bone formation within the defect was delayed with AM around the defect, by 12 weeks, defect BVs were equivalent. Torsional stiffness was significantly reduced with AM but was equivalent to that of intact bone. Collagen + AM resulted in the formation of dense fibrous tissue and mineralized tissue, while the collagen group contained primarily mineralized tissue surrounded by marrow-like structures. Especially in conjunction with high doses of growth factor delivered via collagen sponge, these findings suggest AM may be effective as an overlay adjacent to bone healing sites to spatially direct bone regeneration and minimize heterotopic ossification.