Samuel S. Murray

BMP induced inflammation: a comparison of rhBMP-7 and rhBMP-2.

Concern has been raised because of reports of inflammatory swelling following the use of recombinant human bone morphogenetic protein‐2 (rhBMP‐2) and recombinant human bone morphogenetic protein‐7 (rhBMP‐7). The purpose of this study is to compare the inflammatory action of rhBMP‐7 with those of rhBMP‐2. ELISA assays (IL‐6, TNF‐α) were used to measure the cytokine response to different concentrations of rhBMP‐7 and ‐2. Recombinant human BMP‐7 was absorbed into absorbable collagen sponges and different amounts were implanted either subcutaneously (SC) or intramuscularly (IM) into the backs of rats. Using MRI and MIPAV software, we measured the degree of soft tissue edema at 3 h and at 2, 4, and 7 days postoperatively. After sacrificing rats on day 7 the inflammatory zone and mass were measured and the tissue examined histologically. Soft tissue edema after rhBMP‐7 and rhBMP‐2 implantation was dose‐dependent and peaked at 3 h for the subcutaneous implants and at 2 days for the intramuscular implants. RhBMP‐7 was associated with a significantly smaller soft tissue edema volume than was rhBMP‐2 only at the highest dose (20 µg/ml). Both rhBMP‐2 and rhBMP‐7 triggered dose‐dependent inflammatory reactions. Compared to rhBMP‐2, rhBMP‐7 is associated with somewhat smaller soft tissue edema volumes. Although rhBMP‐7 is associated with an inflammatory reaction leading to soft tissue edema, at high doses this response is significantly less than that seen with rhBMP‐2. Our animal model can be used to test materials that could ameliorate this reaction.

Effects of dietary restriction on total body, femoral, and vertebral bone in SENCAR, C57BL/6, and DBA/2 mice

Dietary restriction (DR) increases the life span and retards aging, in part, by limiting free radical generation and oxidative damage. DR also reduces body mass, a major determinant of bone mass across the life span. We tested the hypothesis that DR has its most beneficial effects on bone in mouse strains with high free radical generation (sensitive to carcinogenesis [SENCAR] > C57 > DBA) versus the hypothesis that bone mass at weight-bearing sites is determined by body mass in DR and ad libitum (AL)-fed mice. Male mice of each strain were killed at 10 weeks of age (t(0)) or randomized to an AL-fed or 30% DR feeding regimen for 6 months. Food consumption by AL-fed mice was measured daily, and DR mice received 70% of the amount of food consumed by their respective AL-fed mice the previous day. Body fat (%) and bone mineral density (BMD) and content (BMC) were determined by PIXImus densitometry. There were strain-dependent effects on body mass, crown-to-rump length, percent body fat, and total body, femoral, and vertebral BMD and BMC under all conditions. SENCAR mice were heavier, longer, had larger bones, and generally exhibited higher total body, femoral, and vertebral BMC and BMD than C57 and DBA mice. DR had beneficial effects on BMD and BMC in the vertebrae of the SENCAR mouse model of high free radical generation and in the obese, diabetes-prone C57 mouse model of high end-stage protein glycation. DR DBA and SENCAR mice had lower femoral BMDs and BMCs than their respective AL-fed controls. Regression analysis confirmed linear relationships between total and lean body mass and total body and femoral BMDs and BMCs, suggesting that physiologic adaptation to a lower body mass accounts for the lower femoral bone mineral values observed in DR versus AL-fed mice. Thus, both hypotheses are, at least, partially valid. DR is beneficial in the trabeculae-rich vertebrae of animal models of high oxidant stress, and total/lean body mass determines BMD and BMC in the weight-bearing femur in DR and AL-fed mice.

The Adjunctive Effect of a Binding Peptide on Bone Morphogenetic Protein Enhanced Bone Healing in a Rodent Model of Spinal Fusion

Study Design. A prospective 8-week interventional trial employing a rat model of spinal fusion to test the effect on bone morphogenetic protein binding peptide (BBP) on rhBMP-2 induced bone healing. Objectives. To determine if the addition of BBP to the collagen sponges used as a carrier for rhBMP-2 reduces the amount of rhBMP-2 required to achieve a satisfactory clinical outcome. Summary of Background Data. Bone morphogenetic proteins (BMPs) although effective in promoting osseous growth and spinal fusion have limitations in their extensive use because of higher costs and possible adverse effects including ectopic bone formation and local inflammatory reaction, particularly in the cervical spine. Methods. Posterolateral intertransverse process spinal fusion at L4–L5 was performed in Lewis rats. Two doses of BBP (500 &mgr;g, and 1000 &mgr;g) were tested with or without “low dose” (1 &mgr;g) rhBMP-2 and the results were compared with the low dose (1 &mgr;g) rhBMP-2. Fusion was evaluated by radiology, histology, and manual palpation tests. Results. Radiology revealed significant earlier fusion with 1000 &mgr;g BBP + 1 &mgr;g BMP-2 combination when compared with low dose BMP-2 (1 &mgr;g) only (P < 0.05). Manual palpation and histology at eighth week revealed higher rate of fusion with the same combination with a nearly significant difference (P = 0.057). Conclusion. Specific growth factor binding agents, such as BBP, can be compounded into carriers used in fusion procedures to decrease the dosage of BMP and possibly decrease the side effects which are most likely dose-related. This may also decrease costs and improve clinical outcomes.

Bone morphogenetic protein binding peptide mechanism and enhancement of osteogenic protein-1 induced bone healing.

Study Design. In vitro and in vivo evaluation of BBP interactions with BMP. Objective. To explore bone morphogenetic protein-binding peptide (BBP)s mechanism of action, investigate an extended repertoire for BBP applications, and evaluate the usefulness of BBP as a surgical adjuvant when used with recombinant human osteogenic protein-1 (rhOP-1). Summary of Background Data. Bone morphogenetic proteins (BMPs) are osteoinductive proteins that provide a potential alternative to autograft. Their utility is limited by cost, and potential dose-dependent risks, such as local inflammatory reactions and ectopic bone formation. BBP, a cyclized synthetic peptide, avidly binds recombinant human BMP-2(rhBMP-2) and has been shown to accelerate and enhance its osteogenic qualities. Methods. BBP binding with 4 growth factors from the transforming growth factor -beta family were assessed using surface plasmon resonance. The in vivo retention of rhBMP-2 was quantified by comparing the percentage of retained [125I]-labeled rhBMP-2 in absorbable collagen sponge implants with or without BBP at 1, 3, and 7 days postimplantation. The adjunctive effect of BBP with rhOP-1-induced bone growth was evaluated by comparing time to fusion and fusion rates in a rodent posterolateral fusion model with 2 different doses of rhOP-1 with or without BBP. Results. BBP bound all 4 growth factors with an intermediate affinity. The in vivo retention of rhBMP-2 alone ranged from about 40% on day 1 to about 30% on day 7, whereas, the retention of rhBMP-2 in the presence of BBP was about 85% on day 1 and about 55% on day 7. The addition of BBP to rhOP-1 resulted in significantly earlier and greater fusion rates than achieved with rhOP-1 alone. Conclusion. The mechanism of the BBP enhanced osteoinductive properties of BMPs involves the binding and retention of the growth factor, resulting in a prolonged exposure of BMP to the desired fusion site. The use of BBP in conjunction with BMPs may prove to provide satisfactory fusion outcomes, while reducing the costs and side effects associated with BMP use.

Full‐length bovine spp24 [spp24 (24‐203)] inhibits BMP‐2 induced bone formation

Secreted phosphoprotein 24 kDa (spp24) is a bone matrix protein. It contains a TGF‐β receptor II homology 1 (TRH1) domain. A cyclic, synthetic 19 amino acid peptide (bone morphogenetic protein binding peptide or BBP) based on the sequence of the TRH1 domain enhances BMP‐2 induced osteogenesis. Many observations suggest that different size forms of this protein have very different effects (inhibiting or enhancing) on BMP‐2 induced osteogenesis. Using the stable recombinant Met(His)6‐tagged secretory form of full‐length (fl) bovine spp24 [Met(His)6‐spp24 (residues 24–203)] and transgenic (TG) mice expressing fl bovine spp24 (residues 1–203), we have demonstrated that spp24 inhibits BMP‐2 induced bone formation. The effects of Met(His)6‐spp24 (24–203) were determined in the ectopic bone‐forming bioassay in male mice. Implantation of 5 µg of BMP‐2 stimulated bone formation, assessed densitometrically as bone area and mineral content. When Met(His)6‐spp24 (24–203) was implanted with BMP‐2, it elicited a dose‐dependent decrease in BMP‐2‐medicated ectopic bone formation. When added at a 50‐fold excess (w/w), Met(His)6‐spp24 (24–203) completely ablated the effects of BMP‐2, while addition of a 10‐fold excess had no effect. Constitutive expression of fl bovine spp24 (1–203) under the control of the osteocalcin promoter in TG female mice reduced femoral and vertebral bone mineral density at 3 months of age and reduced femoral BMD at 8 months of age, but had no effects in male mice, which can exhibit less osteocalcin‐promoter driven gene transcription than females. Histomorphometric analysis demonstrated that bone volume and trabecular thickness were lower in TG female mice at 3 months of age than in sex‐ and age‐matched wild type (WT) controls. Thus, fl spp24 and its secretory isoform (Met(His)6‐spp24 [24–203]), which contain a BMP‐binding or TRH1 motif, inhibit ectopic bone formation in male mice and adversely affects BMD and histological parameters related to bone mass and formation in female mice expressing the human transgene. Under these conditions, fl spp24 acts as a BMP antagonist in vivo.

E64d, a membrane-permeable cysteine protease inhibitor, attenuates the effects of parathyroid hormone on osteoblasts in vitro

Parathyroid hormone (PTH) activates calpains I and II (calcium-activated papain-like proteases) and stimulates the synthesis and secretion of cathepsin B (a lysosomal cysteine protease) in osteoblastic cells. Anabolic doses of PTH also stimulate osteoprogenitor cell proliferation and differentiation into mature, fully functional osteoblasts capable of elaborating bone matrix, whereas catabolic doses of PTH stimulate calcium mobilization and matrix turnover. Previous investigations in other cell types have demonstrated that calcium-activated calpains play a major role in regulating proliferation and differentiation by catalyzing limited regulatory proteolysis of nuclear proteins, transcription factors, and enzymes. We tested the hypothesis that inhibition of intracellular cysteine proteases such as the calpains will ablate PTH-mediated osteoblast proliferation and differentiation, two fundamental indices of bone anabolism. A brief preincubation with the membrane-permeable, irreversible cysteine protease inhibitor E64d (10 micrograms/mL) before short-term PTH treatment blunted PTH-induced cell proliferation in subconfluent cultures and also attenuated proliferation and inhibited differentiation in longer-term confluent cultures. This confirms the hypothesis that cysteine proteases such as the calpains are important in mediating the proliferative and prodifferentiating or anabolic effects of PTH on MC3T3-E1 cells in culture. Immunofluorescent localization demonstrated that calpain I, calpain II, and calpastatin (the endogenous calpain inhibitor) are abundant and widely distributed within actively proliferating MC3T3-E1 preosteoblasts. Since the calpains are active and stable at neutral intracellular pH levels in osteoblasts, whereas cathepsins are not, our results support a role for these calcium-activated regulatory proteases in mediating the anabolic effects of PTH in bone.

Recombinant Expression, Isolation, and Proteolysis of Extracellular Matrix-Secreted Phosphoprotein-24 kDa

Secreted phosphoprotein-24 kDa (spp24) is an extracellular matrix protein first cloned from bone. Bovine spp24 is transcribed as a 203 amino acid residue protein that undergoes cleavage of a secretory peptide to form the mature protein (spp24, residues 24 to 203). While not osteogenic itself, spp24 is degraded to a pro-osteogenic protein, spp18.5, in bone. Both spp18.5 and spp24 contain a cyclic TRH1 (TGF-beta receptor II homology-1) domain similar to that found in the receptor itself and in fetuin. A synthetic peptide corresponding to the TRH1 domain of spp18.5 and spp24 specifically binds BMP-2 and enhances the rate and magnitude of BMP-2-induced ectopic bone formation in vivo. The parental protein, spp24, exhibits a high affinity for bone and mineral complexes, but its abundance there is low, suggesting that it is rapidly degraded. The availability of recombinant spp24 and its degradation products would facilitate the elucidation of their structure:function relationships. We describe here the expression of His6-tagged bovine spp24 (residues 24 to 203) in E. coli, its purification by high-resolution IMAC (immobilized metal affinity chromatography), and the characterization of the full-length recombinant 21.5 kDa protein and its two major 16 kDa and 14.5 kDa degradation products (spp24, residues 24 to 157, and spp24, residues 24 to 143) by mass spectroscopy. The recombinant spp24 protein was resistant to proteolysis by MC3T3-E1 osteoblastic cell extracts in the absence of calcium; however, in the presence of 4 mM Ca, it can undergo essentially complete proteolysis to small peptides, bypassing the 16 kDa and 14.5 kDa intermediates. This confirms the proteolytic susceptibility of spp24. It also suggests that the levels of spp24 in bone may be regulated, in part, by calcium-dependent proteolysis mediated by osteoblastic cells.

Bone morphogenetic protein–2 activity is regulated by secreted phosphoprotein–24 kd, an extracellular pseudoreceptor, the gene for which maps to a region of the human genome important for bone quality

The material properties of bone are the sum of the complex and interrelated anabolic and catabolic processes that modulate formation and turnover. The 2q33-37 region of the human genome contains quantitative trait loci important in determining the broadband ultrasound attenuation (an index of trabecular microarchitecture, bone elasticity, and susceptibility to fracture) of the calcaneus, but no genes of significance to bone metabolism have been identified in this domain. Secreted phosphoprotein-24 kd (SPP24 or SPP2) is a novel and relatively poorly characterized growth hormone-regulated gene that maps to 2q37. The purpose of this review is to summarize the status of research related to spp24 and how it regulates bone morphogenetic protein (BMP) bioactivity in bone. SPP24 codes for an extracellular matrix protein that contains a high-affinity BMP-2-binding transforming growth factor-beta receptor II homology 1 loop similar to those identified in fetuin and the receptor itself. SPP24 is transcribed primarily in the liver and bone. High levels of spp24 (a hydroxyapatite-binding protein) are found in bone, and small amounts are found in fetuin-mineral complexes. Full-length secretory spp24 inhibits ectopic bone formation, and overexpression of spp24 reduces murine bone mass and density. Spp24 is extremely labile to proteolysis, a process that regulates its bioactivity in vivo. For example, an 18.5-kd degradation product of spp24, designated spp18.5, is pro-osteogenic. A synthetic cyclized Cys(1)-to-Cys(19) disulfide-bonded peptide (BMP binding peptide) corresponding to the transforming growth factor-beta receptor II homology 1 domain of spp24 and spp18.5 binds BMP-2 and increases the rate and magnitude of BMP-2-mediated ectopic bone formation. Thus, the mechanism of action of spp18.5 and spp24 may be to regulate the local bioavailability of BMP cytokines. SPP24 is regulated by growth hormone and 3 major families of transcription factors (nuclear factor of activated T cells, CCAAT/enhancer-binding protein, Cut/Cux/CCAAT displacement protein) that regulate mesenchymal cell proliferation, embryonic patterning, and terminal differentiation. The gene contains at least 2 single nucleotide polymorphisms. Given its mechanism of action and sequence variability, SPP24 may be an interesting candidate for future studies of the genetic regulation of bone mass, particularly during periods of BMP-mediated endochondral bone growth, development, and fracture healing.

Identification of the molecular chaperone alpha B-crystallin in demineralized bone powder and osteoblast-like cells.

Bone is subjected to a variety of physiological, as well as cell‐deforming biomechanical stresses, including hydrostatic compression and fluid flow. However, little is known about the molecular mechanisms that protect bone cells from mechanical, ischemic, or oxidative damage. Crystallins are 20 kD heat shock proteins that function as molecular chaperones. We tested the hypothesis that alpha B‐crystallin (αB‐crystallin), the most widely expressed vertebrate crystallin, is present in bone and osteoblast‐like cells. Noncollagenous proteins (NCPs) were extracted from human demineralized bone matrix with 4 M guanidine HCl containing 0.5 M CaCl2 and protease inhibitors, defatted, dialyzed against 0.2% (v/v) Triton X‐100 in 100 mM Tris‐HCI (pH 7.2) and water, centrifuged, and lyophilized. The NCPs were separated by 2D IEF/SDS‐PAGE. The two most abundant 20 kD spots, with apparent pIs of 7.85 and 7.42 in urea gels, were excised, subjected to matrix‐assisted laser desorption ionization/time‐of‐flight mass spectrometry, and identified as αB‐crystallins. Indirect immunofluorescence localized αB‐crystallin to the interphase nucleus, cytoskeleton and cytoplasm of proliferating MC3T3‐El mouse osteoblast‐like cells, as well as the cytoskeleton and cytoplasm of confluent cells. In conclusion, αB‐crystallin is present in bone and osteoblast‐like cells. We hypothesize that αB‐crystallin may play a role in protecting the osteoblast cytoskeleton from mechanical stress and may be important in modulating nuclear or cellular functions, such as transcription or apoptosis, as observed in other tissues.

Effects of the bone morphogenetic protein binding protein spp24 (secreted phosphoprotein 24 kD) on the growth of human lung cancer cells.

Bone morphogenetic proteins (BMPs) and transforming growth factor‐beta (TGF‐β) contribute to the growth of some skeletal metastases through autocrine stimulation. Secreted phosphoprotein 24 kDa (spp24) has been shown to bind to both BMP‐2 and TGF‐β and to markedly inhibit the osteogenic properties of rhBMP‐2. We hypothesized that the addition of spp24 would sequester autocrine growth factors (especially BMP‐2) and reduce tumor growth in a system (A549 human non‐small cell lung cancer cell line) where autocrine stimulation by BMP‐2 is known to be important. A549 cells were injected into two sites (subcutaneous and intraosseus) in SCID mice with and without the co‐injection of BMP‐2 and spp24. Tumor growth after 8 weeks was assessed through gross examination, radiological imaging, and histological analysis. Spp24 attenuated the tumor growth enhancing effects of rhBMP‐2 and reduced the tumor growth when added to tumor cells that were not treated with BMP‐2. We conclude that spp24 can reduce A549 cell tumor growth in both soft tissue and intraosseus environments. We hypothesize that the mechanism for this inhibition is interruption of autocrine stimulation through the sequestration of BMP‐2. Spp24 can be developed into a therapeutic agent that can be employed in clinical situations where the inhibitions of BMPs and related proteins is advantageous.