Elsa J. Brochmann

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.

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.

BMP Stimulation of Alkaline Phosphatase Activity in Pluripotent Mouse C2C12 Cells is Inhibited by Dermatopontin, One of the Most Abundant Low Molecular Weight Proteins in Demineralized Bone Matrix

Demineralized bone matrix (DBM) is a complex mixture of osteoinductive bone morphogenetic proteins (BMPs), as well as BMP-binding proteins that regulate BMP bioactivity and localization. Our aim was to use modern proteomic methods to identify additional BMP-binding proteins in DBM, with initial emphasis on the most abundant. Relatively large, water-soluble noncollagenous proteins (NCPs) were preferentially extracted from DBM with alkalinized urea. The insoluble residue, which contained the BMP activity, was extracted with GuHCl/CaCl2, dialyzed versus citrate, defatted, resuspended in GuHCl, dialyzed sequentially against Triton X-100 and water, pelleted, and lyophilized. The proteins in this pellet were fractionated by hydroxyapatite affinity chromatography. Proteins that copurified with BMP bioactivity were separated by SDS-PAGE. Distinct bands were excised, and the proteins in them were reduced and alkylated, digested with trypsin, eluted, and subjected to MALDI/ToF MS (matrix-assisted laser-desorption ionization time-of-flight mass spectrometry). Computer-assisted peptide fingerprint analysis of the MS profiles was used to identify C-terminal lysine-6-oxidase; dermatopontin (DPT); histones H2A2, H2A3, and H2B; and trace amounts of γ-actin. DPT is a 22-kDa, tyrosine-rich acidic matrix protein not previously recognized to be among the most abundant small proteins to copurify with BMP bioactivity in DBM. We tested the effects of DPT on BMP-2 stimulation of alkaline phosphatase (ALP) activity in C2C12 cells. BMP-2 stimulated ALP activity in C2C12 cells by 6.2-fold above basal levels. DPT alone had no effect on ALP activity in C2C12 cells. When added with BMP-2, DPT blocked 40% of the stimulatory effect of BMP-2 on ALP activity in C2C12 cells. DPT is an abundant protein in DBM, and it can inhibit the stimulatory effects of BMP-2 on ALP activity in C2C12 cells.

Alkali-Urea Extraction of Demineralized Bone Matrix Removes Noggin, an Inhibitor of Bone Morphogenetic Proteins

Demineralized bone matrix (DBM) and native bone morphogenetic protein (nBMP) are complex mixtures of non-collagenous bone proteins. These mixtures contain many of the BMPs that are available as recombinant molecules. Information regarding the presence in these materials of molecules that may affect the availability and activity of the BMPs is very limited. We have devised a simple chemical extraction of DBM using alkali-urea that produces a water soluble extractate that inhibits the osteogenic activity of DBM. We have demonstrated the presence of noggin, an extracellular BMP ligand antagonist, in this material. We conclude that differential chemical extraction may be a useful means of removing inhibitory molecules from DBM and nBMP.

Carboxy terminus of secreted phosphoprotein-24 kDa (spp24) is essential for full inhibition of BMP-2 activity

Secreted phosphoprotein‐24 kDa (spp24) is a bone morphogenetic protein (BMP)‐binding protein isolated from bone. It exists in a number of size forms and is hypothesized to function as a BMP latency protein and/or a “slow release” mechanism for BMPs involved in bone turnover and repair. We have examined the hypothesis that proteolytic modification of the C‐terminus of spp24 affects its BMP‐2–binding properties and bioactivity in the BMP‐2–stimulated ectopic bone forming bioassay. Three different size forms of recombinant spp24 that correspond to predicted 18.1 kDa, 16.0 kDa, and 14.5 kDa proteolytic products were compared to full‐length (fl) spp24. One of these forms (spp18.1) we hypothesize to be the protein which Urist initially, but apparently inaccurately, called “BMP.” Only full‐length spp24 completely inhibited BMP‐2–induced bone formation. The 18.1 kDa truncated isoform of spp24 which we hypothesize to be Urists protein did not. The inhibitory capacity of the proteins was correlated with their kinetic constants, assessed by surface plasmon resonance. At the highest, inhibitory, dose of spp24 and its derivatives, kd (“stability”) best predicted the extent of ectopic bone formation whereas at the lowest dose, which was not inhibitory, ka (“recognition”) best predicted the extent of ectopic bone formation. We conclude that proteolytic processing of spp24 affects the interaction of this protein with BMP‐2 and this affects the function of the protein.