Secreted phosphoprotein 24 kD (Spp24) inhibits the growth of human osteosarcoma through the BMP-2/Smad signaling pathway.

Autocrine stimulation of tumor cells is an important mechanism for the growth of skeletal tumors. In tumors that are sensitive, growth factor inhibitors can dramatically reduce tumor growth. In this study, our aim was to investigate the effects of Secreted phosphoprotein 24 kD (Spp24) on the growth of osteosarcoma (OS) cells in the presence and absence of exogenous BMP-2 both in vitro and in vivo. Our study demonstrated that Spp24 inhibited proliferation and promoted apoptosis of OS cells as confirmed by 3-(4,5-dimethylthiazolyl-2)-2,5-diphenyltetrazolium bromide assay and immunohistochemical staining. We found that BMP-2 increased the mobility and invasiveness of tumor cells in vitro whereas Spp24 inhibited both of these processes alone and in the presence of exogenous BMP-2. Phosphorylation of Smad1/5/8 and Smad8 gene expression was enhanced by treatment with BMP-2 but inhibited by treatment with Spp24. Subcutaneous and intratibial tumor models in nude mice demonstrated that BMP-2 promoted OS growth in vivo, while Spp24 significantly inhibited tumor growth. We conclude that the BMP-2/Smad signaling pathway is involved in the pathogenesis of OS growth and that Spp24 inhibits the growth of human OS induced by BMP-2 both in vitro and in vivo. Interruption of Smad signaling and increased apoptosis appear to be the primary mechanisms involved. These results confirm the potential of Spp24 as a therapeutic agent for the treatment of OS and other skeletal tumors.

Bone morphogenetic protein-2 promotes osteosarcoma growth by promoting epithelial-mesenchymal transition (EMT) through the Wnt/ß-catenin signaling pathway.

The correlation between BMP-2 and osteosarcoma growth has gained increased interest in the recent years, however, there is still no consensus. In this study, we tested the effects of BMP-2 on osteosarcoma cells through both in vitro and in vivo experiments. The effect of BMP-2 on the proliferation, migration and invasion of osteosarcoma cells was tested in vitro. Subcutaneous and intratibial tumor models were used for the in vivo experiments in nude mice. The effects of BMP-2 on EMT of osteosarcoma cells and the Wnt/ß-catenin signaling pathway were also tested using a variety of biochemical methods. In vitro tests did not show a significant effect of BMP-2 on tumor cell proliferation. However, BMP-2 increased the mobility of tumor cells and the invasion assay demonstrated that BMP-2 promoted invasion of osteosarcoma cells in vitro. In vivo animal study showed that BMP-2 dramatically enhanced tumor growth. We also found that BMP-2 induced EMT of osteosarcoma cells. The expression levels of Axin2 and Dkk-1 were both down regulated by BMP-2 treatment, while ß-catenin, c-myc and Cyclin-D1 were all upregulated. The expression of Wnt3α and p-GSK-3ß were also significantly upregulated indicating that the Wnt/ß-catenin signaling pathway was activated during the EMT of osteosarcoma driven by BMP-2. From this study, we can conclude that BMP-2 significantly promotes growth of osteosarcoma cells (143B, MG63), and enhances mobility and invasiveness of tumor cells as demonstrated in vitro. The underlying mechanism might be that BMP-2 promotes EMT of osteosarcoma through the Wnt/ß-catenin signaling pathway. © 2019 Orthopaedic Research Society. Published by Wiley Periodicals, Inc. J Orthop Res 37:1638-1648, 2019.

Bone morphogenetic protein-2 and tumor growth: Diverse effects and possibilities for therapy.

Concern regarding safety with respect to the clinical use of human bone morphogenetic protein-2 (BMP-2) has become an increasingly controversial topic. The role of BMP-2 in carcinogenesis is of particular concern. Although there have been many studies of this topic, the results have been contradictory and confusing. We conducted a systematic review of articles that are relevant to the relationship or effect of BMP-2 on all types of tumors and a total of 97 articles were included. Studies reported in these articles were classified into three major types: "expression studies", "in vitro studies", and "in vivo studies". An obvious pattern was that those works that hypothesize an inhibitory effect for BMP-2 most often examined only the proliferative properties of the tumor cells. This subset of studies also contained an extraordinary number of contradictory findings which made drawing a reliable general conclusion impossible. In general, we support a pro-tumorigenesis role for BMP-2 based on the data from these in vitro cell studies and in vivo animal studies, however, more clinical studies should be carried out to help make a firm conclusion.

Secreted phosphoprotein 24 kD (Spp24) inhibits growth of human pancreatic cancer cells caused by BMP-2.

The emerging role of bone morphogenetic proteins (BMPs) in the initiation and progression of multiple cancers has drawn great attention in cancer research. In this study, we report that BMP-2 can promote the proliferation of the pancreatic tumor cell line, PANC-1. Secreted phosphoprotein 24 kD (Spp24), a BMP binding protein, did not affect the proliferation of the cells but promoted the apoptosis of the cells in vitro. In a xeneograft tumor model using PANC-1 cells, BMP-2 dramatically promoted tumor growth, while Spp24 not only abolished the effect of BMP-2, but also dramatically induced tumor shrinking when used alone. Activation of Smad1/5/8 participated in this process as demonstrated by immunohistochemical staining of phosphorylated Smad 1/5/8. We conclude that Spp24 can be developed into a therapeutic agent that could be employed in clinical situations where the inhibition of BMPs and related proteins is advantageous.

A carboxy terminal BMP/TGF-ß binding site in secreted phosphoprotein 24 kD independently affects BMP-2 activity.

Secreted phosphoprotein 24 kD (spp24) is a bone matrix protein isolated during attempts to identify osteogenic proteins. It is not osteogenic but performs other important roles in the regulation of bone metabolism, at least in part, by binding to and affecting the activity of members of the BMP/TGF-ß family of cytokines. Spp24 exists in a number of forms that preserve the N-terminus and are truncated at the C-terminus. The hypothesized cytokine binding domain is present within the cystatin domain which is preserved in all of the N-terminal products. In this report, we describe a C-terminal fragment that is distinct from the cystatin domain and which independently binds to BMP-2 and TGF-ß. This fragment inhibited BMP-2 activity in an ectopic bone forming assay. A shorter C-terminal product did not inhibit BMP-2 activity but improved bone quality induced by BMP-2 and produced increased calcium deposition outside of bone. Spp24 has been used to develop several potential therapeutic proteins. These results provide more information on the function of spp24 and provide other materials that can be exploited for clinical interventions.

Secreted phosphoprotein 24 kD inhibits nerve root inflammation induced by bone morphogenetic protein-2.

BACKGROUND CONTEXT: Bone morphogenetic protein-2 (BMP-2) has been used to successfully promote spine fusion, but side-effects including nerve inflammation have been observed. PURPOSE: To investigate the direct neurotoxic effects of BMP-2 and test the hypotheses that the use of BMP binding proteins, such as secreted phosphoprotein 24 kD (Spp24), can reduce or eliminate these effects. STUDY DESIGN: In vitro experiments and in vivo analysis in a rodent model. METHODS: In vitro, dorsal root ganglion cells were cultured in the presence of BMP-2 with and without Spp24 and calcitonin gene-related peptide and Substance P, markers of neuroinflammation, were measured by immunohistochemistry. In vivo, rats underwent a left-sided laminotomy at L5 to expose the S1 nerve root and were randomized into four different groups according to the intervention at the laminotomy site: collagen sponge only (no BMP-2 or Spp24), BMP-2 in a collagen sponge only, BMP-2 in a collagen sponge+an empty collagen sponge to act as a barrier, and BMP-2 in a collagen sponge+Spp24 in a collagen sponge to act as a barrier. Functional evaluation was done using the Basso, Beattie, and Bresnahan scale and immunohistochemical analyses were performed using calcitonin gene-related peptide and Substance P staining. RESULTS: The neuroinflammatory effects of BMP-2 in vitro were ameliorated by the addition of Spp24. Similarly, in vivo, Spp24 reduced the expression of markers on neuroinflammation in animals treated with BMP-2 and also improved the function after BMP-2 administration. CONCLUSIONS: These results confirm that BMP binding proteins have great potential as adjuvant therapies to limit BMP-2 related side-effects in spine surgery.

Secreted phosphoprotein 24 kD (Spp24) and Spp14 affect TGF-ß induced bone formation differently.

Transforming growth factor-ß (TGF-ß) and bone morphogenetic proteins (BMPs) have opposing but complementary functions in directing bone growth, repair, and turnover. Both are found in the bone matrix. Proteins that bind to and affect the activity of these growth factors will determine the relative abundance of the growth factors and, therefore, regulate bone formation. Secreted phosphoprotein 24 kD (Spp24) is a bone matrix protein that has been demonstrated to bind to and affect the activity of BMPs. The arginine-rich carboxy terminus of Spp24 is proteolytically processed to produce three other predictable truncation products (Spp18.1, Spp16.0, and Spp14.5). In this work, we report that kinetic data obtained by surface plasmon resonance demonstrate that Spp24 and the three C-terminal truncation products all bind to TGF-ß1 and TGF-ß2 with a similar but somewhat less affinity than they bind BMP-2; that, as in the case of BMP-2, the full-length (FL) form of Spp24 binds TGF-ß with greater affinity than do the truncation products; that FL-Spp24 inhibits TGF-ß2 induced bone formation in vivo, but Spp14.5 does not; and that co-administration of FL-Spp24 or Spp14.5 with TGF-ß2 in vivo is associated with a reduction in the amount of cartilage, relative to new bone, present at the site of injection. This finding is consistent with the observation that low-dose TGF-ß administration in vivo is associated with greater bone formation than high-dose TGF-ß administration, and suggests that one function of Spp24 and its truncation products is to down-regulate local TGF-ß activity or availability during bone growth and development. The similarities and differences of the interactions between Spp24 proteins and TGF-ß compared to the interaction of the Spp24 proteins and BMPs have significant implications with respect to the regulation of bone metabolism and with respect to engineering therapeutic proteins for skeletal disorders.

In vivo inhibition of bone morphogenetic protein-2 on breast cancer cell growth.

STUDY DESIGN: In vitro and in vivo study. OBJECTIVE: To evaluate the role of recombinant human bone morphogenetic protein-2 (rhBMP2) on breast cancer cell (MDA-MB-231 cells) growth. SUMMARY OF BACKGROUND DATA: Bone morphogenetic proteins (BMPs) are expressed in a variety of human carcinoma cell lines and are known to promote tumor invasion and metastasis. However, their roles in tumor progression have not been fully clarified. In addition, there is no in vivo study regarding the inhibitory effect of BMP2 on breast cancer cell proliferation. METHOD: Cell proliferation was determined by BrdU incorporation assay and flow cytometry. BMP2 signal transduction pathways were estimated on Western blot. Fifteen animals were divided into 2 groups; 1 (control = 5) was breast cancer cells alone, while the other (experiment = 5) was rhBMP2 + breast cancer cells. Cancer cells were injected into 2 sites (subcutaneous and femur) of nude mice with or without BMP2. Tumor size was determined by direct measurements for subcutaneous tumor formation and by femur radiographs. Histological and immunohistochemical analyses were performed. RESULTS: RhBMP2 inhibited the proliferation of MDA-MB-231 cells in vitro. Inhibition was associated with changes in both the Smad and Wnt signaling pathways and was ultimately mediated through effects on various cell cycle proteins. Furthermore, rhBMP2 inhibited the growth of MDA-MB-231 cells injected both subcutaneously and intrafemorally. CONCLUSION: In this model using human breast adenocarcinoma cell line, rhBMP2 has no stimulatory effect of tumor growth. Therefore, we can provide the basic science data to support the utilization in the management of patients with spine tumor in the future.

Bone morphogenetic protein-binding peptide reduces the inflammatory response to recombinant human bone morphogenetic protein-2 and recombinant human bone morphogenetic protein-7 in a rodent model of soft-tissue inflammation.

BACKGROUND CONTEXT: Bone morphogenetic protein (BMP)-2 and BMP-7 are used to enhance bone formation in spine surgery, but the use of these materials is associated with side effects including inflammation, especially in the soft tissues of the neck. Bone morphogenetic protein-binding peptide (BBP) binds BMP-2 and BMP-7 and imparts a "slow-release" property to collagen carrier. PURPOSE: To test the hypothesis that the addition of BBP will reduce the soft-tissue inflammation induced by the implantation of BMP-2 and BMP-7 on a collagen sponge. STUDY DESIGN/SETTING: Prospective in vivo rodent model of inflammation. METHODS: We implanted six different materials absorbed onto collagen sponges: absorbable collagen sponge (ACS) alone; BBP alone; recombinant human bone morphogenetic protein (rhBMP)-2 alone; rhBMP-2 plus BBP; rhBMP-7 alone; and rhBMP-7 plus BBP. Sponges were implanted bilaterally (subcutaneously [SC] and intramuscularly [IM]) into the backs of rats. Using magnetic resonance imaging, inflammation was assessed in terms of soft-tissue edema volume at 3 hours and at 2, 4, and 7 days. The animal subjects were killed on Day 7, and the dimensions of the inflammatory mass were measured manually in the case of SC tissue and those of the inflammatory zone were determined subsequently by microscopic examination in the case of muscle. RESULTS: Both the SC and the IM soft-tissue edema volumes in the rhBMP-2 plus BBP and the rhBMP-7 plus BBP groups were significantly lower than those observed in the rhBMP-2 alone and rhBMP-7 alone groups. The edema volume associated with BBP alone was greater than that associated with ACS alone but less than that associated with the other treatment groups. The measurements of inflammatory masses and zone yielded similar results. CONCLUSIONS: Bone morphogenetic protein-binding peptide may reduce the inflammatory response associated with the use of rhBMP-2 and rhBMP-7 in a rodent model of inflammation and in a form that has previously been shown to enhance the activity of BMPs. These preliminary studies suggest that BBP may have the potential to be used in the future to improve healing and reduce soft-tissue swelling in surgical applications of BMPs.

Full-length spp24, but not its 18.5-kDa proteolytic fragment, inhibits bone-healing in a rodent model of spine fusion.

BACKGROUND: Growth factors like bone morphogenetic protein (BMP) are used as bone-graft substitutes to enhance bone growth in clinical situations. However, adverse reactions have been associated with BMP use. We developed a synthetic adjuvant therapy based on the sequence of a BMP-binding protein, secreted phosphoprotein-24 (spp24), which enhances the effects of BMPs and ameliorates the adverse reactions. Our hypothesis is that a natural proteolytic fragment of spp24 is identical to an osteogenic protein previously described independently by two investigators. To test this hypothesis, spp24 and a truncated form of spp24 were separately implanted with recombinant human BMP-2 (rhBMP-2) in a rodent model of spine fusion. METHODS: Two isoforms of spp24 were constructed with use of DNA recombinant technology. Spp24 with or without rhBMP-2 were added to collagen sponges and implanted bilaterally between L4 and L5 transverse processes. Radiographs were made biweekly, and spines were explanted after eight weeks. Gross evaluation, microquantitative computed tomography study, and histological analysis were performed to evaluate bone growth. RESULTS: Animals that received full-length spp24 and rhBMP-2 exhibited a complete obliteration of bone growth, while animals with the truncated form in combination with rhBMP-2 exhibited a mild inhibition to bone growth, with bone area measured from radiographs. Manual assessment and gross evaluation of all spines confirmed the results obtained from the bone-area measurements. Microquantitative computed tomography provided three-dimensional visual images of representative specimens, while histological staining of spine tissue displayed cellular evidence of bone formation. CONCLUSIONS: Results from this investigation confirm that the various isoforms of spp24 affect the bone-healing activity of rhBMP-2 in the rat spine fusion model. Thus, proteolytic modification of this protein is a likely mechanism for the regulation of BMP availability in the physiological environment. Future studies will define the roles of these proteins in controlling the activity of BMPs and other members of the transforming growth factor-beta family of cytokines. This information will increase the understanding of normal bone-healing, allowing for the engineering of more effective orthopaedic treatment.

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-ß) 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-ß 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.

Alanine-scanning mutations of the BMP-binding domain of recombinant secretory bovine spp24 affect cytokine binding.

Secreted phosphoprotein 24 kDa (spp24) is a bone morphogenetic protein (BMP)/transforming growth factor-ß cytokine-binding protein. The spp24 BMP-2-binding/transforming growth factor receptor II homology-1 (TRH1) domain is a highly conserved N-to-C terminally disulfide-bonded 19-amino acid residue loop similar to those in fetuin and the BMP receptor II. TRH1 domains exhibit a characteristic BTB or ß-pleated sheet/turn/ß-pleated sheet secondary structure. Our objective was to identify amino acid residues in the spp24 TRH1 domain that bind BMP-2, starting with the nine invariant mammalian residues. Alanine scanning (substitution of Ala for a native residue) was conducted for Cys(110), Arg(111), Ser(112), Thr(113), Val(114), Ser(117), Val(121), Val(124), and Cys(128) of recombinant bovine spp24 (residues 24-203). Binding to rhBMP-2 was assessed by surface plasmon resonance, and the equilibrium binding constants were calculated assuming 1:1 binding between spp24 or its mutants and rhBMP-2, so that affinity = K(D) = k(d)/k(a). Replacing Arg(111) (a positively charged basic residue), polar residues Thr(113) and Ser(117), and the nonpolar Cys(128) with Ala had little effect on BMP-2 binding. Replacing Val(114) or Val(121) with Ala increased binding affinity, whereas replacing Cys(110), Ser(112), Val(124), or both Cys(110) and Cys(128) with Ala decreased it. The kinetics of spp24 binding to BMP-2 can be manipulated by replacing invariant TRH1 residues. Decreasing the relative degree of hydrophobicity in the ß-pleated sheet secondary structural motif of the TRH1 domain by replacing key Val residues with Ala increased the affinity for BMP-2 whereas altering the composition of the α-helical turn did not. Thus, the ß-pleated sheets play a greater role in BMP-2 binding than the α-helical turn.

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.

Absence of bone morphogenetic protein-2 in human breast milk after spinal surgery.

BACKGROUND CONTEXT: Bone morphogenetic protein-2 (BMP-2) has been used extensively in recent years to enhance the process of spinal fusion. However, numerous side effects have been reported. This raises concerns regarding the safety of using this material in human beings. There are no published reports, that we are aware of, that demonstrate detectable levels of BMP-2 in human milk. PURPOSE: The purpose of this study was to determine the presence/absence of recombinant human bone morphogenetic protein-2 (rhBMP-2) in milk samples taken from a patient who underwent spinal surgery using rhBMP-2 shortly after delivering a child. STUDY DESIGN/SETTING: The study design comprises a case report dealing with analyzing milk from a patient who underwent spinal surgery using rhBMP-2 shortly after delivering a child. METHODS: We report the analysis of milk samples from a patient who underwent spinal surgery using rhBMP-2 shortly after delivering a child to determine the presence/absence of rhBMP-2. The milk samples were taken before and after the spinal surgery. Enzyme-linked immunosorbent assays were performed repeatedly to the samples. RESULTS: The standard curve for the assay had an r(2) value of 0.9853 indicating an acceptable degree of statistical reliability. The dose range for the standard curve was 62.5 to 2,000 pg/mL. None of the samples had an optical density greater than that of the lowest standard. CONCLUSIONS: No rhBMP-2 was detected in human milk in this single case report. Although this result seemed promising, it did not eliminate the concern regarding the potential risk of rhBMP-2 on child development. We still need further studies including more cases to verify this conclusion.

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 Urist's 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, k(d) ("stability") best predicted the extent of ectopic bone formation whereas at the lowest dose, which was not inhibitory, k(a) ("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.

Enhanced effects of BMP-binding peptide combined with recombinant human BMP-2 on the healing of a rodent segmental femoral defect.

BMP-binding peptide (BBP) enhances the osteogenic activity of recombinant human bone morphogenetic protein-2 (rhBMP-2), but the mechanism underlying the enhancement remains unclear. We aimed to elucidate the potential enhanced efficacy of BBP using critical-sized segmental femoral bone defects in rats. Seventy defects in seven groups of rats were filled with various amounts (0, 2, 5, and 10 microg) of rhBMP-2 with or without 1000 microg BBP. Radiographs were obtained after 4 and 8 weeks. The animals were euthanized at 8 weeks, and femoral specimens were assessed manually, evaluated for bone volume using microcomputed tomography, and subjected to histological or biomechanical analysis. Although 10 microg rhBMP-2 yielded consistent results in terms of bone healing and quality of bone repair across the segmental defect, lower doses of rhBMP-2 failed to induce satisfactory bone healing. However, the combined administration of lower doses of rhBMP-2 and BBP induced the formation of significantly large amounts of bone. Our results suggest that the combined administration of rhBMP-2 and BBP facilitates bone healing and has potential clinical applications.

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.

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 microg, and 1000 microg) were tested with or without "low dose" (1 microg) rhBMP-2 and the results were compared with the low dose (1 microg) rhBMP-2. Fusion was evaluated by radiology, histology, and manual palpation tests. RESULTS: Radiology revealed significant earlier fusion with 1000 microg BBP + 1 microg BMP-2 combination when compared with low dose BMP-2 (1 microg) 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.

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-beta 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 microg 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.

A statistical model to allow the phasing out of the animal testing of demineralised bone matrix products.

Demineralised bone matrix (DBM) products are complex mixtures of proteins known to influence bone growth, turnover, and repair. They are used extensively in orthopaedic surgery, and are bioassayed in vivo prior to being used in clinical applications. Many factors contribute to the osteogenic potency of DBM, but the relative contributions of these factors, as well as the possibility of interactive effects, are not completely defined. The "gold standard" measure of the therapeutic value of DBM, the in vivo assay for ectopic bone formation, is costly, time-consuming, and involves the use of numerous animal subjects. We have measured the levels of five growth factors released by the collagenase digestion of DBM, and statistically related these levels with osteogenic potency as determined by a standard in vivo model, in order to determine which value or combination of values of growth factors best predict osteogenic activity. We conclude that the level of BMP-2 is the best single predictor of osteogenic potency, and that adding the values of other growth factors only minimally increases the predictive power of the BMP-2 measurement. A small, but significant, interactive effect between BMP-2 and BMP-7 was demonstrated. We present a statistical model based on growth factor (e.g. BMP-2) analysis that best predicts the in vivo assay score for DBM. This model allows the investigator to predict which lots of DBM are likely to exhibit in vivo bioactivity and which are not, thus reducing the need to conduct in vivo testing of insufficiently active lots of DBM. This model uses cut-point analysis to allow the user to assign an estimate of acceptable uncertainty with respect to the "gold standard" test. This procedure will significantly reduce the number of animal subjects used to test DBM products.