Therapeutics for enhancement of spinal fusion: A mini review.

OBJECTIVE: With the advances in biological technologies over the past 20 years, a number of new therapies to promote bone healing have been introduced. Particularly in the spinal surgery field, more unprecedented biological therapeutics become available to enhance spinal fusion success rate along with advanced instrumentation approaches. Yet surgeons may not have been well informed about their safety and efficacy profiles in order to improve clinical practices. Therefore there is a need to summarize the evidence and bring the latest progress to surgeons for better clinical services for patients. METHODS: We comprehensively reviewed the literatures in regard to the biological therapeutics for enhancement of spinal fusion published in the last two decades. RESULTS: Autograft bone is still the gold standard for bone grafting in spinal fusion surgery due to its good osteoconductive, osteoinductive, and osteogenic abilities. Accumulating evidence suggests that adding rhBMPs in combination with autograft effectively promotes the fusion rate and improves surgical outcomes. However, the stimulating effect on spinal fusion of other growth factors, including PDGF, VEGF, TGF-beta, and FGF, is not convincing, while Nell-1 and activin A exhibited preliminary efficacy. In terms of systemic therapeutic approaches, the osteoporosis drug Teriparatide has played a positive role in promoting bone healing after spinal surgery, while new medications such as denosumab and sclerostin antibodies still need further validation. Currently, other treatment, such as controlled-release formulations and carriers, are being studied for better releasing profile and the administration convenience of the active ingredients. CONCLUSION: As the world's population continues to grow older, the number of spinal fusion cases grows substantially due to increased surgical needs for spinal degenerative disease (SDD). Critical advancements in biological therapeutics that promote spinal fusion have brought better clinical outcomes to patients lately. With the accumulation of higher-level evidence, the safety and efficacy of present and emerging products are becoming more evident. These emerging therapeutics will shift the landscape of perioperative therapy for the enhancement of spinal fusion.

A novel auditory ossicles membrane and the development of conductive hearing loss in Dmp1-null mice.

Genetic mouse models are widely used for understanding human diseases but we know much less about the anatomical structure of the auditory ossicles in the mouse than we do about human ossicles. Furthermore, current studies have mainly focused on disease conditions such as osteomalacia and rickets in patients with hypophosphatemia rickets, although the reason that these patients develop late-onset hearing loss is unknown. In this study, we first analyzed Dmp1 lac Z knock-in auditory ossicles (in which the blue reporter is used to trace DMP1 expression in osteocytes) using X-gal staining and discovered a novel bony membrane surrounding the mouse malleus. This finding was further confirmed by 3-D micro-CT, X-ray, and alizarin red stained images. We speculate that this unique structure amplifies and facilitates sound wave transmissions in two ways: increasing the contact surface between the eardrum and malleus and accelerating the sound transmission due to its mineral content. Next, we documented a progressive deterioration in the Dmp1-null auditory ossicle structures using multiple imaging techniques. The auditory brainstem response test demonstrated a conductive hearing loss in the adult Dmp1-null mice. This finding may help to explain in part why patients with DMP1 mutations develop late-onset hearing loss, and supports the critical role of DMP1 in maintaining the integrity of the auditory ossicles and its bony membrane.

Sclerostin antibody (Scl-Ab) improves osteomalacia phenotype in dentin matrix protein 1(Dmp1) knockout mice with little impact on serum levels of phosphorus and FGF23.

Unlike treatments for most rickets, the treatment using 1,25-(OH)2 vitamin D3 has little efficacy on patients with hypophosphatemic rickets, a set of rare genetic diseases. Thus, understanding the local cause for osteomalacia in hypophosphatemic rickets and developing an effective treatment to restore mineralization in this rare disease has been a longstanding goal in medicine. Here, we used Dmp1 knockout (KO) mice (whose mutations led to the same type of autosomal recessive hypophosphatemic rickets in humans) as the model in which the monoclonal antibody of sclerostin (Scl-Ab) was tested in two age groups for 8weeks: the prevention group (starting at age 4weeks) and the treatment group (starting at age 12weeks). Applications of Scl-Ab greatly improved the osteomalacia phenotype (>15%) and the biomechanical properties (3-point bending, ~60%) in the treated long-bone group. Our studies not only showed improvement of the osteomalacia in the alveolar bone, which has the highest bone metabolism rate, as well as the long bone phenotypes in treated mice. All these improvements attributed to the use of Scl-Ab are independent of the change in serum levels of phosphorus and FGF23, since Scl-Ab had little efficacy on those parameters. Finally, we propose a model to explain how Scl-Ab can improve the Dmp1 KO osteomalacia phenotype, in which the sclerostin level is already low.

The in vivo role of DMP-1 and serum phosphate on bone mineral composition.

Human DMP1 mutations or Dmp1-null (KO) mice display hypophosphatemia rickets, suggesting a causative role of low phosphate (P) in development of osteomalacia. To address the direct contribution of P to the in vivo bone mineralization we analyzed the properties of femurs obtained from Dmp1 null mice and wild type (WT) mice under a normal or high phosphorous (HiP) diet using combined assays, including histological examination, micro computed tomography (µCT), X-ray absorption near edge structure (XANES) spectroscopy and Raman spectroscopy. Histology and XANES indicate that WT mice have phosphate coordinated with Ca in the form of hydroxyapatite and tricalcium phosphate, while the KO mice have poorly coordinated soluble phosphates in their structure in both the normal and HiP diets. Raman spectroscopy and XANES indicate a higher carbonate/phosphate ratio and a low mineral/matrix ratio in the osteoid clusters in the KO femurs, which was only partially improved by HiP diets. Thus, we conclude that the hypophosphatemia induced osteomalacia phenotype in Dmp1 KO mice is contributed by at least two factors: the low Pi level and the DMP1 local function in mineralization.

Big endothelin changes the cellular miRNA environment in TMOb osteoblasts and increases mineralization.

INTRODUCTION: Endothelin (ET1) promotes the growth of osteoblastic breast and prostate cancer metastases. Conversion of big ET1 to mature ET1, catalyzed primarily by endothelin converting enzyme 1 (ECE1), is necessary for ET1's biological activity. We previously identified the Ece1, locus as a positional candidate gene for a pleiotropic quantitative trait locus affecting femoral size, shape, mineralization, and biomechanical performance. METHODS: We exposed TMOb osteoblasts continuously to 25 ng/ml big ET1. Cells were grown for 6 days in growth medium and then switched to mineralization medium for an additional 15 days with or without big ET1, by which time the TMOb cells form mineralized nodules. We quantified mineralization by alizarin red staining and analyzed levels of miRNAs known to affect osteogenesis. Micro RNA 126-3p was identified by search as a potential regulator of sclerostin (SOST) translation. RESULTS: TMOb cells exposed to big ET1 showed greater mineralization than control cells. Big ET1 repressed miRNAs targeting transcripts of osteogenic proteins. Big ET1 increased expression of miRNAs that target transcripts of proteins that inhibit osteogenesis. Big ET1 increased expression of 126-3p 121-fold versus control. To begin to assess the effect of big ET1 on SOST production we analyzed both SOST transcription and protein production with and without the presence of big ET1 demonstrating that transcription and translation were uncoupled. CONCLUSION: Our data show that big ET1 signaling promotes mineralization. Moreover, the results suggest that big ET1's osteogenic effects are potentially mediated through changes in miRNA expression, a previously unrecognized big ET1 osteogenic mechanism.

Osteocyte regulation of phosphate homeostasis and bone mineralization underlies the pathophysiology of the heritable disorders of rickets and osteomalacia.

Although recent studies have established that osteocytes function as secretory cells that regulate phosphate metabolism, the biomolecular mechanism(s) underlying these effects remain incompletely defined. However, investigations focusing on the pathogenesis of X-linked hypophosphatemia (XLH), autosomal dominant hypophosphatemic rickets (ADHR), and autosomal recessive hypophosphatemic rickets (ARHR), heritable disorders characterized by abnormal renal phosphate wasting and bone mineralization, have clearly implicated FGF23 as a central factor in osteocytes underlying renal phosphate wasting, documented new molecular pathways regulating FGF23 production, and revealed complementary abnormalities in osteocytes that regulate bone mineralization. The seminal observations leading to these discoveries were the following: 1) mutations in FGF23 cause ADHR by limiting cleavage of the bioactive intact molecule, at a subtilisin-like protein convertase (SPC) site, resulting in increased circulating FGF23 levels and hypophosphatemia; 2) mutations in DMP1 cause ARHR, not only by increasing serum FGF23, albeit by enhanced production and not limited cleavage, but also by limiting production of the active DMP1 component, the C-terminal fragment, resulting in dysregulated production of DKK1 and ß-catenin, which contributes to impaired bone mineralization; and 3) mutations in PHEX cause XLH both by altering FGF23 proteolysis and production and causing dysregulated production of DKK1 and ß-catenin, similar to abnormalities in ADHR and ARHR, but secondary to different central pathophysiological events. These discoveries indicate that ADHR, XLH, and ARHR represent three related heritable hypophosphatemic diseases that arise from mutations in, or dysregulation of, a single common gene product, FGF23 and, in ARHR and XLH, complimentary DMP1 and PHEX directed events that contribute to abnormal bone mineralization.

The rescue of dentin matrix protein 1 (DMP1)-deficient tooth defects by the transgenic expression of dentin sialophosphoprotein (DSPP) indicates that DSPP is a downstream effector molecule of DMP1 in dentinogenesis.

Dentin matrix protein 1 (DMP1) and dentin sialophosphoprotein (DSPP) are essential for the formation of dentin. Previous in vitro studies have indicated that DMP1 might regulate the expression of DSPP during dentinogenesis. To examine whether DMP1 controls dentinogenesis through the regulation of DSPP in vivo, we cross-bred transgenic mice expressing normal DSPP driven by a 3.6-kb rat Col1a1 promoter with Dmp1 KO mice to generate mice expressing the DSPP transgene in the Dmp1 KO genetic background (referred to as "Dmp1 KO/DSPP Tg mice"). We used morphological, histological, and biochemical techniques to characterize the dentin and alveolar bone of Dmp1 KO/DSPP Tg mice compared with Dmp1 KO and wild-type mice. Our analyses showed that the expression of endogenous DSPP was remarkably reduced in the Dmp1 KO mice. Furthermore, the transgenic expression of DSPP rescued the tooth and alveolar bone defects of the Dmp1 KO mice. In addition, our in vitro analyses showed that DMP1 and its 57-kDa C-terminal fragment significantly up-regulated the Dspp promoter activities in a mesenchymal cell line. In contrast, the expression of DMP1 was not altered in the Dspp KO mice. These results provide strong evidence that DSPP is a downstream effector molecule that mediates the roles of DMP1 in dentinogenesis.

Protective roles of DMP1 in high phosphate homeostasis.

PURPOSE: Dmp1 (dentin matrix protein1) null mice (Dmp1(-/-)) display hypophosphatemic rickets with a sharp increase in fibroblast growth factor 23 (FGF23). Disruption of Klotho (the obligatory co-receptor of FGF23) results in hyperphosphatemia with ectopic calcifications formed in blood vessels and kidneys. To determine the role of DMP1 in both a hyperphosphatemic environment and within the ectopic calcifications, we created Dmp1/Klotho compound deficient (Dmp1(-/-)kl/kl) mice. PROCEDURES: A combination of TUNEL, immunohistochemistry, TRAP, von Kossa, micro CT, bone histomorphometry, serum biochemistry and Scanning Electron Microscopy techniques were used to analyze the changes in blood vessels, kidney and bone for wild type control, Dmp1(-/-), Klotho deficient (kl/kl) and Dmp1(-/-)kl/kl animals. FINDINGS: Interestingly, Dmp1(-/-)kl/kl mice show a dramatic improvement of rickets and an identical serum biochemical phenotype to kl/kl mice (extremely high FGF23, hyperphosphatemia and reduced parathyroid hormone (PTH) levels). Unexpectedly, Dmp1(-/-)kl/kl mice presented elevated levels of apoptosis in osteocytes, endothelial and vascular smooth muscle cells in small and large blood vessels, and within the kidney as well as dramatic increase in ectopic calcification in all these tissues, as compared to kl/kl. CONCLUSION: These findings suggest that DMP1 has an anti-apoptotic role in hyperphosphatemia. Discovering this novel protective role of DMP1 may have clinical relevance in protecting the cells from apoptosis in high-phosphate environments as observed in chronic kidney disease (CKD).

Inactivation of a novel FGF23 regulator, FAM20C, leads to hypophosphatemic rickets in mice.

Family with sequence similarity 20,-member C (FAM20C) is highly expressed in the mineralized tissues of mammals. Genetic studies showed that the loss-of-function mutations in FAM20C were associated with human lethal osteosclerotic bone dysplasia (Raine Syndrome), implying an inhibitory role of this molecule in bone formation. However, in vitro gain- and loss-of-function studies suggested that FAM20C promotes the differentiation and mineralization of mouse mesenchymal cells and odontoblasts. Recently, we generated Fam20c conditional knockout (cKO) mice in which Fam20c was globally inactivated (by crossbreeding with Sox2-Cre mice) or inactivated specifically in the mineralized tissues (by crossbreeding with 3.6 kb Col 1a1-Cre mice). Fam20c transgenic mice were also generated and crossbred with Fam20c cKO mice to introduce the transgene in the knockout background. In vitro gain- and loss-of-function were examined by adding recombinant FAM20C to MC3T3-E1 cells and by lentiviral shRNA-mediated knockdown of FAM20C in human and mouse osteogenic cell lines. Surprisingly, both the global and mineralized tissue-specific cKO mice developed hypophosphatemic rickets (but not osteosclerosis), along with a significant downregulation of osteoblast differentiation markers and a dramatic elevation of fibroblast growth factor 23 (FGF23) in the serum and bone. The mice expressing the Fam20c transgene in the wild-type background showed no abnormalities, while the expression of the Fam20c transgene fully rescued the skeletal defects in the cKO mice. Recombinant FAM20C promoted the differentiation and mineralization of MC3T3-E1 cells. Knockdown of FAM20C led to a remarkable downregulation of DMP1, along with a significant upregulation of FGF23 in both human and mouse osteogenic cell lines. These results indicate that FAM20C is a bone formation "promoter" but not an "inhibitor" in mouse osteogenesis. We conclude that FAM20C may regulate osteogenesis through its direct role in facilitating osteoblast differentiation and its systemic regulation of phosphate homeostasis via the mediation of FGF23.

Unique roles of phosphorus in endochondral bone formation and osteocyte maturation.

The mechanisms by which inorganic phosphate (P(i)) homeostasis controls bone biology are poorly understood. Here we used Dmp1 null mice, a hypophosphatemic rickets/osteomalacia model, combined with a metatarsal organ culture and an application of neutralizing fibroblast growth factor 23 (FGF-23) antibodies to gain insight into the roles of P(i) in bone biology. We showed (1) that abnormal bone remodeling in Dmp1 null mice is due to reduced osteoclast number, which is secondary to a reduced ratio of RANKL/OPG expressed by osteoclast supporting cells and (2) that osteoblast extracellular matrix mineralization, growth plate maturation, secondary ossification center formation, and osteoblast differentiation are phosphate-dependent. Finally, a working hypothesis is proposed to explain how phosphate and DMP1 control osteocyte maturation.

The biological function of DMP-1 in osteocyte maturation is mediated by its 57-kDa C-terminal fragment.

Dentin matrix protein 1 (DMP-1) is a key molecule in controlling osteocyte formation and phosphate homeostasis. Based on observations that full-length DMP-1 is not found in bone, but only cleaved fragments of 37 and 57 kDa are present, and in view of the finding that mutations in the 57-kDa fragment result in disease, we hypothesized that the 57-kDa C-terminal fragment is the functional domain of DMP-1. To test this hypothesis, a 3.6-kb type I collagen promoter was used to express this 57-kDa C-terminal fragment for comparison with full-length DMP-1 in Dmp1 null osteoblasts/osteocytes. Not only did expression of the full-length DMP-1 in bone cells fully rescue the skeletal abnormalities of Dmp1 null mice, but the 57-kDa fragment also had similar results. This included rescue of growth plate defects, osteomalacia, abnormal osteocyte maturation, and the abnormal osteocyte lacunocanalicular system. In addition, the abnormal fibroblast growth factor 23 (FGF-23) expression in osteocytes, elevated circulating FGF-23 levels, and hypophosphatemia were rescued. These results show that the 57-kDa C-terminal fragment is the functional domain of DMP-1 that controls osteocyte maturation and phosphate metabolism.

Aberrant Phex function in osteoblasts and osteocytes alone underlies murine X-linked hypophosphatemia.

Patients with X-linked hypophosphatemia (XLH) and the hyp-mouse, a model of XLH characterized by a deletion in the Phex gene, manifest hypophosphatemia, renal phosphate wasting, and rickets/osteomalacia. Cloning of the PHEX/Phex gene and mutations in affected patients and hyp-mice established that alterations in PHEX/Phex expression underlie XLH. Although PHEX/Phex expression occurs primarily in osteoblast lineage cells, transgenic Phex expression in hyp-mouse osteoblasts fails to rescue the phenotype, suggesting that Phex expression at other sites underlies XLH. To establish whether abnormal Phex in osteoblasts and/or osteocytes alone generates the HYP phenotype, we created mice with a global Phex knockout (Cre-PhexDeltaflox/y mice) and conditional osteocalcin-promoted (OC-promoted) Phex inactivation in osteoblasts and osteocytes (OC-Cre-PhexDeltaflox/y). Serum phosphorus levels in Cre-PhexDeltaflox/y, OC-Cre-PhexDeltaflox/y, and hyp-mice were lower than those in normal mice. Kidney cell membrane phosphate transport in Cre-PhexDeltaflox/y, OC-Cre-PhexDeltaflox/y, and hyp-mice was likewise reduced compared with that in normal mice. Abnormal renal phosphate transport in Cre-PhexDeltaflox/y and OC-Cre-PhexDeltaflox/y mice was associated with increased bone production and serum FGF-23 levels and decreased kidney membrane type IIa sodium phosphate cotransporter protein, as was the case in hyp-mice. In addition, Cre-PhexDeltaflox/y, OC-Cre-PhexDeltaflox/y, and hyp-mice manifested comparable osteomalacia. These data provide evidence that aberrant Phex function in osteoblasts and/or osteocytes alone is sufficient to underlie the hyp-mouse phenotype.

Loss of DMP1 causes rickets and osteomalacia and identifies a role for osteocytes in mineral metabolism.

The osteocyte, a terminally differentiated cell comprising 90%-95% of all bone cells, may have multiple functions, including acting as a mechanosensor in bone (re)modeling. Dentin matrix protein 1 (encoded by DMP1) is highly expressed in osteocytes and, when deleted in mice, results in a hypomineralized bone phenotype. We investigated the potential for this gene not only to direct skeletal mineralization but also to regulate phosphate (P(i)) homeostasis. Both Dmp1-null mice and individuals with a newly identified disorder, autosomal recessive hypophosphatemic rickets, manifest rickets and osteomalacia with isolated renal phosphate-wasting associated with elevated fibroblast growth factor 23 (FGF23) levels and normocalciuria. Mutational analyses showed that autosomal recessive hypophosphatemic rickets family carried a mutation affecting the DMP1 start codon, and a second family carried a 7-bp deletion disrupting the highly conserved DMP1 C terminus. Mechanistic studies using Dmp1-null mice demonstrated that absence of DMP1 results in defective osteocyte maturation and increased FGF23 expression, leading to pathological changes in bone mineralization. Our findings suggest a bone-renal axis that is central to guiding proper mineral metabolism.