Long-term clinical data have shown that reconstruction using <em>bone</em> allografts provide adequate function after extensive tumor surgery. Complications such as nonunion of allograft-host interface, infection, and allograft fracture often require major revision surgeries. Allograft fractures usually do not induce the same repair process that is seen in normal fracture healing. The authors did an experimental study to test whether <em>bone</em> <em>morphogenetic</em> <em>protein</em>-2 can induce and achieve osseous repair in an allograft osteotomy model. Recombinant human <em>bone</em> <em>morphogenetic</em> <em>protein</em>-2 was applied at femoral intercalary allograft osteotomy sites in 20 rats. Forty additional rats served as controls (carrier alone and sham). Specimens in all groups were examined histologically and radiographically at 4 and 8 weeks. Specimens in the control groups showed only fibrosis by 8 weeks. In contrast, none of <em>10</em> specimens in the experimental group showed radiographic union at 8 weeks. New <em>bone</em> formation and integration with underlying allografts were seen in the experimental group as early as 4 weeks. These data suggest that fracture repair in the allograft <em>bone</em> can be triggered by a biologic regulator that is expressed during normal fracture healing.

Tumor necrosis factor-alpha (TNF-alpha) is considered a promoter of <em>bone</em> resorption and a suppressor of osteogenesis, whereas <em>bone</em> <em>morphogenetic</em> <em>protein</em> (BMP) is a promoter of <em>bone</em> formation. In the present study, the osteogenic potential of the medullary cavity after <em>bone</em> marrow ablation was evaluated in association with the pattern of BMP, <em>bone</em> <em>morphogenetic</em> <em>protein</em> receptor (BMPR), TNF-alpha, and tumor necrosis factor receptor (TNFR) expression. Immunostaining, in situ hybridization, and TRAP staining were performed following marrow ablation. By day 4, BMP-4 mRNA was detected by in situ hybridization in growing undifferentiated cells and, on day 7, the osteoblastic cells that covered abundant woven <em>bone</em> also showed evidence of BMP-4 expression. BMPR-IA and BMPR-II were immunolocalized from days 4 to <em>10</em> after ablation, and became negative on days 21 and 28. At <em>10</em> days postablation, osteoclasts were revealed by TRAP staining. TNF-alpha expression disappeared transiently after ablation and then reappeared on day 7, predominantly in osteoblastic cells. On days 7, <em>10</em>, and 14, immunostaining for TNFR-I was observed in osteoblasts lining the woven <em>bone</em> and later disappeared. No evidence of TNFR-II staining was observed on osteoblastic cells throughout the reaction. From day 14, newly formed <em>bone</em> decreased in quantity and was replaced by hematopoietic cells and, by day 28, the <em>bone</em> marrow had regenerated to its original state. This study suggests that TNF-alpha is produced and secreted by the osteoblast and acts on these cells in an autocrine manner to suppress osteoblastic function. TNF-alpha may also play a role in the recruitment of osteoclasts because TRAP-positive osteoclasts appeared after TNF-alpha expression.

BACKGROUND

Considerable debate exists regarding the incidence of persistent pain from the iliac crest bone graft (ICBG) harvest site. Different study designs have led to a variety of reported rates.

OBJECTIVE

The purpose of this study was to determine the incidence and severity of bone graft site pain after iliac crest harvest.

METHODS

Cross-sectional.

METHODS

One hundred and twelve patients, who had a posterior lumbar fusion, seen at a tertiary spine center for a routine postoperative visit.

METHODS

Numeric rating scales (0-10) for pain over lower back, right, and left posterior iliac crests.

METHODS

An independent investigator, not directly involved in the care of the patient and unaware of the type of bone graft used in the fusion, examined the patient for tenderness over the surgical site as well as the left and right posterior iliac crest. After the examination, data on the source of grafting material, complications during harvest, and backfilling of the graft site defect were collected from the medical records. The patients were then classified as to whether ICBG was harvested or not. Chi-square test was used to determine any difference in the proportion of iliac crest pain between the bone graft group and no bone graft group. Correlations between body mass index (BMI), time since surgery, and the incidence and severity of bone graft site pain were also determined.

RESULTS

There were 72 women and 40 men with a mean age of 56.6 years (range, 16-84). Mean follow-up was 41 months (range, 6-211 months) with a median of 25 months. Iliac crest bone graft was harvested in 53 (47.3%) patients through the midline incision used for lumbar fusion. In 59 patients (52.7%), recombinant human bone morphogenetic protein-2 was used with no graft harvest. There was no statistically significant difference in the proportion of patients complaining of tenderness over both or either iliac crest between the two groups. Only 10 patients had pain over the same crest from which the graft was harvested. No correlations between number of levels fused, levels fused, BMI, length of follow-up, and the incidence and severity of bone graft site pain were seen.

CONCLUSIONS

The results of this study highlight the difficulty in differentiating pain originating from the graft site versus residual low back pain. The incidence of pain over the iliac crest was similar in patients in which iliac crest was harvested and those in which no graft was harvested.

OBJECTIVE

Tooth eruption is a localized event that requires the expression of certain molecules at precise times to regulate bone resorption and bone formation. Parathyroid hormone-related protein (PTHrP) may be one of those molecules. Although PTHrP is produced in the stellate reticulum (SR) of the tooth and exerts its effect on the adjacent dental follicle, its expression pattern in the SR is unknown. Thus, it was the objectives of this study to determine the chronology of expression of PTHrP, and then to determine its effect on vascular endothelial growth factor (VEGF) expression for osteoclastogenesis and on bone morphogenetic protein-2 (BMP-2) for bone growth.

METHODS

Laser capture microdissection and RT-PCR were used to determine the chronological expression of PTHrP in vivo. In vitro, dental follicle cells were incubated with PTHrP and RT-PCR was conducted to determine its effect on VEGF and BMP-2 gene expression.

RESULTS

PTHrP was maximally expressed at day 7 postnatally in the SR with the level of expression still high at day 9. In vitro, PTHrP upregulated VEGF120 and VEGF164 expression after 4h of incubation with a maximum effect at 6h. PTHrP upregulated BMP-2 gene expression with a maximal effect at 2h.

CONCLUSIONS

Because the secondary burst of osteoclastogenesis needed for eruption occurs around day 10, it is possible that PTHrP is stimulating this osteoclastogenesis by upregulating VEGF. Concurrently, the upregulation of BMP-2 by PTHrP may stimulate bone growth at the base of the bony crypt to promote eruption.

Endochondral ossification implicates chondrocyte signaling as an important factor in directing the osteogenic differentiation of mesenchymal stem cells in vivo. In this study, the osteoinductive capabilities of articular chondrocytes suspended in alginate hydrogels were analyzed via coculture with <em>bone</em> marrow stromal cells (BMSCs). In particular, the effect of chondrocyte coculture time on the mechanism underlying this osteogenic induction was examined. Chondrocytes were suspended in alginate beads and cultured above BMSCs in monolayer. Beads containing chondrocytes were removed after 1, <em>10</em>, or 21 days of coculture. Quantitative reverse transcriptase polymerase chain reaction was used to assess the expression of alkaline phosphatase, <em>bone</em> <em>morphogenetic</em> <em>protein</em>-2, and osteocalcin by BMSCs after days 1, 8, 14, and 21. Calcium deposition was also assayed to characterize the extent of mineralization within cultures. Results indicate that osteogenic differentiation of BMSCs is initiated upon brief exposure to chondrocyte signaling, but requires continued exposure in order to progress fully and maintain an osteoblastic phenotype.

Osteoblast cell adhesion and differentiation on biomaterials are important achievements necessary for implants to be useful in <em>bone</em> regenerative engineering. Recombinant <em>bone</em> <em>morphogenetic</em> <em>proteins</em> (BMPs) have been shown to be important for these processes; however, there are many challenges associated with the widespread use of these <em>proteins</em>. A recent report demonstrated that the small molecule phenamil, a diuretic derivative, was able to induce osteoblast differentiation and mineralization in vitro via the canonical BMP signalling cascade (Park et al., 2009). In this study, the feasibility of using phenamil as a novel biofactor in conjunction with a biodegradable poly(lactide-co-glycolide acid) (PLAGA) polymeric scaffold for engineering <em>bone</em> tissue was evaluated. The in vitro cellular behaviour of osteoblast-like MC3T3-E1 cells cultured on PLAGA scaffolds in the presence of phenamil at <em>10</em> μM were characterized with regard to initial cell adhesion, proliferation, alkaline phosphatase (ALP) activity and matrix mineralization. The results demonstrate that phenamil supported cell proliferation, promoted ALP activity and facilitated matrix mineralization of osteoblast-like MC3T3-E1 cells. Moreover, in this study, we found that phenamil promoted integrin-mediated cell adhesion on PLAGA scaffolds. It was also shown that phenamil encapsulated within porous, microsphere PLAGA scaffolds retained its osteogenic activity upon release. Based on these findings, the small molecule phenamil has the potential to serve as a novel biofactor for the repair and regeneration of <em>bone</em> tissues.

The success of ex vivo viral gene therapy systems for promoting <em>bone</em> formation could be improved through the development of systems to spatially localize gene expression. Towards this goal, we have encapsulated adenovirus-transduced human diploid fetal lung fibroblasts (MRC-5) expressing <em>bone</em> <em>morphogenetic</em> <em>protein</em>-type 2 (BMP-2) within non-degradable poly(ethylene glycol)-diacrylate (PEG-DA) hydrogels and implanted these intramuscularly to promote endochondral <em>bone</em> formation. To optimize BMP-2 secretion, the molecular weight of the polymers and cell densities were varied. Polymers with molecular weights of 6, <em>10</em>, and 20 kDa were used to prepare hydrogels containing 1, 5, or <em>10</em> million transduced cells. The results showed that <em>10</em> million transduced fibroblasts that was the maximum number of cells feasible for encapsulation within PEG-DA <em>10</em> and 20 kDa hydrogels produced the highest amount of secreted BMP-2 <em>protein</em>. Encapsulation of MRC-5 and transduced fibroblasts resulted in 71 and 58% cell viability, respectively. The bioactivity of secreted BMP-2 <em>protein</em> from the hydrogels was confirmed with an alkaline phosphatase assay. Micro-CT of the lower limb muscles of NOD/SCID mice following implantation with hydrogels showed 39.5 +/- 25.0 mm3 mineralized tissue and 31.8 +/- 7.8 mm3 for the cell-injected mice, and the <em>bone</em> was localized to the hydrogel surfaces. Histology revealed <em>bone</em> as well as cartilage for both hydrogel implanted and cell-injected animals.

Clonal cell lines presumably "arrested" at a particular stage of differentiation are useful models to study the processes of differentiation in osteoblasts. UMR-201 is a presumptive preosteoblastic nontransformed rat clonal cell line with a limited life span in culture. Two immortalized cell lines, UMR-201-<em>10</em>A (<em>10</em>A) and UMR-201-<em>10</em>B (<em>10</em>B), were derived from UMR-201 by stable transfection with simian virus (SV) 40 large T antigen. This study compares the growth and profile of gene expression of the immortalized cell lines with those of UMR-201 and UMR-<em>10</em>6-06, a rat clonal cell line with well-defined osteoblast-like phenotypic characteristics. All four cell lines constitutively expressed the mRNA for the gamma, alpha, and beta receptors for retinoic acid (RA), the growth hormone receptor, pro-alpha 1(I) collagen, osteonectin, <em>bone</em> proteoglycan I, and <em>bone</em> <em>morphogenetic</em> <em>proteins</em> (BMP) 1 and 2A. Alkaline phosphatase mRNA was absent in the preosteoblast cell lines but was induced by treatment with <em>10</em>(-6) M RA, which also increased the steady-state levels of mRNA for osteopontin and BMP1. mRNA for matrix gla <em>protein</em> was constitutively present and further induced by RA in UMR-201 and <em>10</em>B only. Messenger RNA for <em>bone</em> sialo<em>protein</em> and <em>bone</em> <em>morphogenetic</em> <em>protein</em> 3 were constitutively expressed in UMR-<em>10</em>6-06 and UMR-201 but absent in the immortalized cell lines. None of the cell lines expressed measurable mRNA for <em>bone</em> gla <em>protein</em> or <em>bone</em> proteoglycan II. <em>10</em>B grew more rapidly than UMR-201, but unlike UMR-201, it was also able to proliferate in serum-free medium and exhibit anchorage-independent growth. In summary, this study identifies novel retinoic acid effects on gene expression in these cells. Differences noted in the expression of mRNAs between UMR-<em>10</em>6-06 and the other cell lines may provide some insight into the sequence of expression of these phenotypic characteristics as osteoblasts differentiate.

<em>Bone</em> <em>morphogenetic</em> <em>proteins</em> (BMPs) are cytokines from the TGF-beta superfamily, with important roles during embryonic development and in the induction of <em>bone</em> and cartilage tissue differentiation in the adult body. In this contribution, we report the expression of recombinant human BMP-4, BMP-9, BMP-<em>10</em>, BMP-11 (or growth differentiation factor-11, GDF-11) and BMP-14 (GDF-5), using Escherichia coli pET-25b vector. BMPs were overexpressed, purified by affinity his-tag chromatography and shown to induce the expression of early markers of <em>bone</em> differentiation (e.g. smad-1, smad-5, runx2/cbfa1, dlx5, osterix, osteopontin, <em>bone</em> sialoprotein and alkaline phosphatase) in C2C12 cells and in human adipose stem cells. The described approach is a promising method for producing large amounts of different recombinant BMPs that show potential for novel biomedical applications.

BACKGROUND

Transforming growth factor-beta (TGF-beta) and bone morphogenetic proteins (BMPs) utilize parallel and related signaling pathways, however the interaction between these pathways in bone remains unclear. TGF-beta inhibition has been previously reported to promote osteogenic differentiation in vitro, suggesting it may have a capacity to augment orthopaedic repair. We have explored this concept using an approach that represents a template for the testing of agents with prospective orthopaedic applications.

METHODS

The effects of BMP-2, TGF-beta1, and the TGF-beta receptor (ALK-4/5/7) inhibitor SB431542 on osteogenic differentiation were tested in the MC3T3-E1 murine pre-osteoblast cell line. Outcome measures included alkaline phosphatase staining, matrix mineralization, osteogenic gene expression (Runx2, Alp, Ocn) and phosphorylation of SMAD transcription factors. Next we examined the effects of SB431542 in two orthopaedic animal models. The first was a marrow ablation model where reaming of the femur leads to new intramedullary bone formation. In a second model, 20 microg rhBMP-2 in a polymer carrier was surgically introduced to the hind limb musculature to produce ectopic bone nodules.

RESULTS

BMP-2 and SB431542 increased the expression of osteogenic markers in vitro, while TGF-beta1 decreased their expression. Both BMP-2 and SB431542 were found to stimulate pSMAD1 and we also observed a non-canonical repression of pSMAD2. In contrast, neither in vivo system was able to provide evidence of improved bone formation or repair with SB431542 treatment. In the marrow ablation model, systemic dosing with up to 10 mg/kg/day SB431542 did not significantly increase reaming-induced bone formation compared to vehicle only controls. In the ectopic bone model, local co-administration of 38 microg or 192 microg SB431542 did not increase bone formation.

CONCLUSIONS

ALK-4/5/7 inhibitors can promote osteogenic differentiation in vitro, but this may not readily translate to in vivo orthopaedic applications.

For the clinical usage of human-derived <em>bones</em>, it is necessary to treat <em>bones</em> to reduce the risk of contamination by microorganisms. <em>Bone</em> <em>morphogenetic</em> <em>protein</em> is vulnerable to chemicals, but shows resistance to thermal heat to 70 degrees C in a short time. In this experiment, crude human <em>bone</em> <em>morphogenetic</em> <em>protein</em> was extracted from heat-treated <em>bones</em> at 60 degrees C for <em>10</em> hours and from nonheated <em>bones</em>. Sodium dodecyl sulfate polyacrylamide gel electrophoresis for these specimens was done. Gelatin capsules containing 5 mg of crude human <em>bone</em> <em>morphogenetic</em> <em>protein</em> extracted from heated and nonheated <em>bones</em> were implanted into thigh muscle pouches of five mice. At 20 days after implantation, the heterotopic <em>bone</em> formation was compared by evaluating the radiographic and histologic analyses. The sodium dodecyl sulfate polyacrylamide gel electrophoresis pattern of the human <em>bone</em> <em>morphogenetic</em> <em>proteins</em> showed five main bands (16, 22, 28, 35, and 67 kDa) that were almost identical. Heterotopic <em>bone</em> formation observed on the radiograph was induced by crude human <em>bone</em> <em>morphogenetic</em> <em>protein</em> from heated <em>bones</em> in a manner similar to that used for nonheated <em>bones</em>. The results from this study show that heat-treated <em>bone</em> preserves osteoinduction.

Hereditary hemorrhagic telangiectasia (HHT) is a highly debilitating and life-threatening genetic vascular disorder arising from endothelial cell (EC) proliferation and hypervascularization, for which no cure exists. Because HHT is caused by loss-of-function mutations in <em>bone</em> <em>morphogenetic</em> <em>protein</em> 9 (BMP9)-ALK1-Smad1/5/8 signaling, interventions aimed at activating this pathway are of therapeutic value. We interrogated the whole-transcriptome in human umbilical vein ECs (HUVECs) and found that ALK1 signaling inhibition was associated with a specific pro-angiogenic gene expression signature, which included a significant elevation of DLL4 expression. By screening the NIH clinical collections of FDA-approved drugs, we identified tacrolimus (FK-506) as the most potent activator of ALK1 signaling in BMP9-challenged C2C12 reporter cells. In HUVECs, tacrolimus activated Smad1/5/8 and opposed the pro-angiogenic gene expression signature associated with ALK1 loss-of-function, by notably reducing Dll4 expression. In these cells, tacrolimus also inhibited Akt and p38 stimulation by vascular endothelial growth factor, a major driver of angiogenesis. In the BMP9/<em>10</em>-immunodepleted postnatal retina-a mouse model of HHT vascular pathology-tacrolimus activated endothelial Smad1/5/8 and prevented the Dll4 overexpression and hypervascularization associated with this model. Finally, tacrolimus stimulated Smad1/5/8 signaling in C2C12 cells expressing BMP9-unresponsive ALK1 HHT mutants and in HHT patient blood outgrowth ECs. Tacrolimus repurposing has therefore therapeutic potential in HHT.

Sib-pair linkage analysis of the quantitative trait, structure, in over 500 Pima Indians indicates that a genetic determinant of governing stature is located on chromosome 20. Analysis of <em>10</em> short tandem repeat polymorphisms localized this linkage to a 3.2cM region that includes D20S98 and D20S66. Using all possible sib-pair combinations, linkage was detected to both stature (P = 0.0001) and to leg length (P = 0.001), but not to sitting height. Single-strand conformational polymorphism analysis of exon 3 of the <em>bone</em> <em>morphogenetic</em> <em>protein</em> 2 (BMP2) gene, a candidate gene in this region, in genomic DNA of 20 of the tallest and 20 of the shortest individuals did not show any consistent differences associated with leg length or height. Sequence analysis of the region encoding the mature <em>protein</em> revealed a single nucleotide substitution, a T to G transversion, not detected by single-strand conformational polymorphism (SSCP) analysis. This transversion results in a conservative amino acid substitution of glycine for valine at codon 80 of BMP2. The frequency of this allele was 0.23 in the sample. No significant differences in height were noted in persons carrying either allele. This indicates that this structural alteration is the mature BMP2 <em>protein</em> does not contribute to the differences in stature observed in the Pima Indians, nor is this structural change in the mature <em>protein</em> likely to be responsible for the linkage observed with stature on chromosome 20.

Fish hatching enzymes are zinc metalloproteases that digest the egg envelope (chorion) at the time of hatching. The crystal structure of zebrafish hatching enzyme 1 (ZHE1) has been solved at 1.<em>10</em> Å resolution. ZHE1 is monomeric, is mitten shaped, and has a cleft at the center of the molecule. ZHE1 consists of three 3(<em>10</em>)-helices, three α-helices, and two β-sheets. The central cleft represents the active site of the enzyme that is crucial for substrate recognition and catalysis. Alanine-scanning mutagenesis of the two substrate peptides has shown that AspP1' contributes the most and that the residues at P4-P2' also contribute to the recognition of the major substrate peptide by ZHE1, whereas GluP3' and the hydrophobic residues at P4-P2, P2', and P5' contribute significantly to the recognition of the minor substrate peptide by ZHE1. Molecular models of these two substrate peptides bound to ZHE1 have been built based on the crystal structure of a transition-state analog inhibitor bound to astacin. In substrate-recognition models, the AspP1' in the major substrate peptide forms a salt bridge with Arg182 of ZHE1, while the GluP3' in the minor substrate peptide instead forms a salt bridge with Arg182. Thus, these two substrate peptides would be differently recognized by ZHE1. The shapes and electrostatic potentials of the substrate-binding clefts of ZHE1 and the structurally similar <em>proteins</em> astacin and <em>bone</em> <em>morphogenetic</em> <em>protein</em> 1 are significantly dissimilar due to different side chains, which would confer their distinctive substrate preferences.

OBJECTIVE

To delineate the dynamic micro-architectures of bone induced by low-dose bone morphogenetic protein (BMP)-2/7 heterodimer in peri-implant bone defects compared to BMP2 and BMP7 homodimer.

METHODS

Peri-implant bone defects (8 mm in diameter, 4 mm in depth) were created surrounding SLA-treated titanium implants (3.1 mm in diameter, 10 mm in length) in minipig's calvaria. We administrated collagen sponges with adsorbed low-dose (30 ng/mm(3) ) BMP2/7 to treat the defects using BMP2, BMP7 or no BMP as controls.2, 3 and 6 weeks after implantation, we adopted micro-computer tomography to evaluate the micro-architectures of new bone using the following parameters: relative bone volume (BV/TV), trabecular number (Tb.N), trabecular thickness (Tb.Th), trabecular separation (Tb.Sp), connectivity density, and structure mode index (SMI). Bone implant contact (BIC) was also revealed histologically.

RESULTS

Consistent with 2 and 3 weeks, after 6 weeks post-operation, BMP2/7 resulted in significantly higher BV/TV (63.033 ± 2.055%) and significantly lower SMI (-4.405 ± 0.500) than BMP2 (BV/TV: 43.133 ± 2.001%; SMI: -0.086 ± 0.041) and BMP7 (BV/TV: 41.467 ± 1.850%; SMI: -0.044 ± 0.016) respectively. Significant differences were also found in Tb.N, Tb.Th and Tb.Sp at all time points. At 2 weeks, BMP2/7 resulted in significantly higher BIC than the controls.

CONCLUSIONS

Low-dose BMP2/7 heterodimer facilitated more rapid bone regeneration in better quality in peri-implant bone defects than BMP2 and BMP7 homodimers.

We identified a gene encoding a novel secreted <em>protein</em> in mice and humans and named it Brorin. Mouse Brorin consists of 324 amino acids with a putative secreted signal sequence at its amino terminus and two cysteine-rich domains in its core region. Positions of <em>10</em> cysteine residues in the domains of Brorin are similar to those in the cysteine-rich domains of members of the Chordin family. However, the amino acid sequence of Brorin is not significantly similar to that of any other member of the Chordin family, indicating that Brorin is a unique member of the family. Mouse Brorin <em>protein</em> produced in cultured cells was efficiently secreted into the culture medium. The <em>protein</em> inhibited the activity of <em>bone</em> <em>morphogenetic</em> <em>protein</em> 2 (BMP2) and BMP6 in mouse preosteoblastic MC3T3-E1 cells. Mouse Brorin was predominantly expressed in neural tissues in embryos and also predominantly expressed in the adult brain. In the brain, the expression was detected in neurons, but not glial cells. The neural tissue-specific expression profile of Brorin is quite distinct from that of any other member of the Chordin family. Brorin <em>protein</em> promoted neurogenesis, but not astrogenesis, in mouse neural precursor cells. The present findings indicate that Brorin is a novel secreted BMP antagonist that potentially plays roles in neural development and functions.

<em>Bone</em> <em>morphogenetic</em> <em>protein</em> (BMP)-2 and transforming growth factor (TGF)-beta1 are multifunctional cytokines both proposed as stimulants for cartilage repair. Thus it is crucial to closely examine and compare their effects on the expression of key markers of the chondrocyte phenotype, at the gene and <em>protein</em> level. In this study, the expression of alpha <em>10</em> and alpha 11 integrin subunits and the IIA/IIB spliced forms of type II procollagen have been monitored for the first time in parallel in the same in vitro model of mouse chondrocyte dedifferentiation/redifferentiation. We demonstrated that TGF-beta1 stimulates the expression of the non-chondrogenic form of type II procollagen, IIA isoform, and of a marker of mesenchymal tissues, i.e. the alpha 11 integrin subunit. On the contrary, BMP-2 stimulates the cartilage-specific form of type II procollagen, IIB isoform, and a specific marker of chondrocytes, i.e. the alpha <em>10</em> integrin subunit. Collectively, our results demonstrate that BMP-2 has a better capability than TGF-beta1 to stimulate chondrocyte redifferentiation and reveal that the relative expressions of type IIB to type IIA procollagens and alpha <em>10</em> to alpha 11 integrin subunits are good markers to define the differentiation state of chondrocytes. In addition, adenoviral expression of Smad6, an inhibitor of BMP canonical Smad signaling, did not affect expression of total type II procollagen or the ratio of type IIA and type IIB isoforms in mouse chondrocytes exposed to BMP-2. This result strongly suggests that signaling pathways other than Smad <em>proteins</em> are involved in the effect of BMP-2 on type II procollagen expression.

Surgical treatment of post-traumatic or neoplastic <em>bone</em> defects often represents a problem in orthopaedic routine. Autologous tissue always stands for the first choice material. The recent therapeutic approaches for tissue repair of <em>bone</em> defects attempt to mimic the natural process of <em>bone</em> repair by delivering a source of cells capable of differentiating into osteoblasts. In this study two different types of human osteoblast cell harvesting were compared in primary cell culture to evaluate the best way to obtain cells for clinical use. Numerous articles describe the characteristics of each one of these systems, but there is no report comparing the influence of these different isolation techniques on cell growth. Cultures from 22 <em>bone</em> specimens obtained from donors were established in two different ways and their proliferation was compared. An enzymatic procedure to extract human osteoblasts (hOBcol) was used in one group; spontaneous cells outgrowth, human osteoblasts (hOBsog) was expected in the other group. Cells of both groups were characterised as osteoblasts by immunohistochemical staining with <em>Bone</em> <em>Morphogenetic</em> <em>Protein</em>-2,4 (BMP-2,4), expression of collagen type I as well as the amount of alkaline phosphatase activity (ALP) detected in the cell cultures. We found that the time needed in primary cultures till confluence was dependent on the age of the donors as well as on the method of cell harvesting. Cells from under 65-year old donors were growing significantly faster by the hOBcol method as compared to hOBsog 20.57 +/- 2.99 days vs. 30.00 +/- 4.36. Cells harvested from donors older than 65 years of age needed 23.88 +/- 2.95 in the hOBcol compared to 34.25 +/- 4.27 days in the other group. In the experimental cultures, after one passage with trypsin/EDTA, there was a significant difference between the two groups. There was an improved cell growth in the hOBsog group found on days 8, 9 and <em>10</em> of cultivation. Immunohistochemical staining as well as ALP activity were similar in both groups. In conclusion this study evaluated an important step for a tissue engineering approach to the repair of <em>bone</em> defects, which may have clinical applications in post-traumatic orthopaedic surgery.

Many investigators are currently studying the use of decellularized tissue allografts from human cadavers as scaffolds onto which patients' cells could be seeded, or as carriers for genetically engineered cells to aid cell transplantation. However, it is difficult to seed cells onto very dense regular connective tissue which has few interstitial spaces. Here, we discuss the development of a chemotactic cell seeding technique using solvent-preserved human meniscus. A chemokinetic response to recombinant human <em>bone</em> <em>morphogenetic</em> <em>protein</em>-2 (rhBMP-2) was observed in a monolayer culture of primary chondrocytes derived from femoral epiphyseal cartilage of 2-day-old rats. The rhBMP-2 significantly increased their migration upto <em>10</em> ng/ml in a dose-dependent manner. When tested with solvent-preserved human meniscus as a scaffold, which has few interstitial spaces, rhBMP-2 was able to induce chondrocytes to migrate into the meniscus. After a 3-week incubation, newly-formed cartilaginous extracellular matrix was synthesized by migrated chondrocytes throughout the meniscus, down to a depth of 3 mm. These findings demonstrate that rhBMP-2 may be a natural chemokinetic factor in vivo, which induces migration of proliferative chondrocytes into the narrow interfibrous spaces. Our results suggest a potential application of rhBMP-2 for the designed distribution of chondrocytes into a scaffold to be used for tissue engineering.

OBJECTIVE

Recombinant human bone morphogenetic protein-2 (rhBMP-2) is a well-known growth factor that can induce robust bone formation, and recent studies have shown that rhBMP-2-induced osteogenesis is closely related to adipogenesis. The aim of the present study was to determine the dose- and time-dependent effects of rhBMP-2 on the osteogenic and adipogenic differentiation of human alveolar bone-derived stromal cells (hABCs) in vivo and in vitro.

METHODS

hABCs were isolated and cultured, and then transplanted using a carrier treated either with or without rhBMP-2 (100 μg/mL) into an ectopic subcutaneous mouse model. Comprehensive histologic and histometric analyses were performed after an 8-wk healing period. To further understand the dose-dependent (0, 10, 50, 200, 500 and 1000 ng/mL) and time-dependent (0, 3, 5, 7 and 14 d) effects of rhBMP-2 on osteogenic and adipogenic differentiation, in vitro osteogenic and adipogenic differentiation of hABCs were evaluated, and the expression of related mRNAs, including those for alkaline phosphatase, osteocalcin, bone sialoprotein, peroxisome-proliferator-activated receptor gamma-2 and lipoprotein lipase, were assessed using quantitative RT-PCR.

RESULTS

rhBMP-2 significantly promoted the osteogenic and adipogenic differentiation of hABCs in vivo, and gradually increased both the osteogenic and adipogenic potential in a dose- and time-dependent manner with minimal deviation in vitro. The expression of osteogenesis- and adipogenesis-associated mRNAs were concomitantly up-regulated by rhBMP-2.

CONCLUSIONS

The findings of the present study showed that rhBMP-2 significantly enhanced the adipogenic as well as the osteogenic potential of hABCs in dose- and time-dependent manner. The control of adipogenic differentiation of hABCs should be considered when regenerating the alveolar bone using rhBMP-2.

BACKGROUND

Reports have identified cells capable of osteogenic differentiation in bone marrow, muscle, and adipose tissues, but there are few direct comparisons of these different cell types. Also, few have investigated the potential connection between a tissue-specific abnormality and cells derived from seemingly unrelated tissues. In this article, the authors compare cells isolated from wild-type rabbits or rabbits with nonsyndromic craniosynostosis, defined as the premature fusion of one or more of the cranial sutures.

METHODS

Cells were derived from bone marrow, adipose, and muscle of 10-day-old wild-type rabbits (n = 17) or from age-matched rabbits with familial nonsyndromic craniosynostosis (n = 18). Cells were stimulated with bone morphogenetic protein-4 (BMP4), and alkaline phosphatase expression and cell proliferation were assessed.

RESULTS

In wild-type rabbits, cells derived from muscle had more alkaline phosphatase activity than cells derived from either adipose or bone marrow. The cells derived from craniosynostotic rabbit bone marrow and muscle were significantly more osteogenic than those derived from wild-type rabbits. Adipose-derived cells demonstrated no significant differences. Although muscle-derived cells were most osteogenic in wild-type rabbits, bone marrow-derived cells were most osteogenic in craniosynostotic rabbits.

CONCLUSIONS

These results suggest that cells from different tissues have different potentials for differentiation. Furthermore, cells derived from rabbits with craniosynostosis were different from cells from wild-type rabbits. Interestingly, cells derived from the craniosynostotic rabbits were not uniformly more responsive compared with wild-type cells, suggesting that specific tissue-derived cells may react differently in individuals with craniosynostosis.

Animal model for heterotopic ossification (HO) induced by Achilles tenotomy in rats has been used in the literature. However, the molecular mechanism remains unclear. Here, we studied <em>bone</em> and cartilage related genes and their possible roles in this model. Thirty rats underwent bilateral midpoint Achilles tenotomy through a posterior approach under aseptic condition. At 3, 5 and <em>10</em> weeks post-operation, X-ray and histological examinations were carried out to investigate the formation of HO. At different phases of HO formation, mRNA levels of transforming growth factor (TGF)-beta1, TGF-beta2, TGF-beta 3, <em>bone</em> <em>morphogenetic</em> <em>proteins</em> (BMP)-2, BMP-4, BMP-7, hypoxia inducible factor (HIF)-1 alpha, Sox9, Runx2, vascular endothelial growth factor (VEGF), aggrecan and collagen type I, II and X were evaluated by real-time RT-PCR. <em>Protein</em> levels of TGF-beta1, TGF-beta2, TGF-beta 3, BMP-2, BMP-4, BMP-7, HIF-1 alpha, Sox9 and Runx2 were also examined by immunohistochemical staining. During the chondrogenic phase, the expressions of HIF-1 alpha and Sox9 were significantly up-regulated. Runx2 expression was significantly up-regulated during osteogenic phase, while HIF-1 alpha and Sox9 expression was significant decreased. TGF-beta1 mRNA levels were rather constant, and the mRNA levels of TGF-beta2, TGF-beta 3 and BMPs were changed throughout HO formation. The presences of the <em>proteins</em> of HIF-1 alpha, Sox9, Runx2, TGF-betas and BMPs within the HO tissues were confirmed by immunohistochemical staining. Our study indicates that HO induced by Achilles tenotomy is by endochondral <em>bone</em> formation, and HIF-1 alpha activation plays an important role during chondrogenesis in this model. Furthermore, the model provides a new experimental system to study endochondral ossification.

BACKGROUND

Glucosamine is an amino-monosaccharide and precursor of glycosaminoglycans, major components of joint cartilage. Glucosamine has been clinically introduced for the treatment of osteoarthritis but the data about its protective role in disease are insufficient. The goal of this study was to investigate the effect of long term administration of glucosamine on bone resorption and remodeling.

METHODS

The effect of glucosamine on bone resorption and remodeling was studied in a model of collagenase-induced osteoarthritis (CIOA). The levels of macrophage-inflammatory protein (MIP)-1α, protein regulated upon activation, normal T-cell expressed, and secreted (RANTES), soluble receptor activator of nuclear factor kappa-B ligand (RANKL), tumor necrosis factor (TNF)-α, and interleukin (IL)-6, 4 and 10 in synovial fluid were measured by enzyme-linked immunosorbent assay (ELISA). Cell populations in synovial extracts and the expression of RANKL, of receptors for TNF-α (TNF-αR) and interferon γ (IFN-γR) on clusters of differentiation (CD) three positive T cells were analyzed by flow cytometry. Transforming growth factor (TGF)-β3, bone morphogenetic protein (BMP)-2, phosphorylated protein mothers against decapentaplegic homolog 2 (pSMAD-2), RANKL and Dickkopf-1 protein (DKK-1) positive staining in CIOA joints were determined by immunohistochemistry.

RESULTS

The administration of glucosamine hydrochloride in CIOA mice inhibited loss of glycosaminoglycans (GAGs) and proteoglycans (PGs) in cartilage, bone erosion and osteophyte formation. It decreased the levels of soluble RANKL and IL-6 and induced IL-10 increase in the CIOA joint fluids. Glucosamine limited the number of CD11b positive Ly6G neutrophils and RANKL positive CD3 T cells in the joint extracts. It suppressed bone resorption via down-regulation of RANKL expression and affected bone remodeling in CIOA by decreasing BMP-2, TGF-β3 and pSMAD-2 expression and up-regulating DKK-1 joint levels.

CONCLUSIONS

Our data suggest that glucosamine hydrochloride inhibits bone resorption through down-regulation of RANKL expression in the joints, via reduction of the number of RANKL positive CD3 T cells and the level of sRANKL in the joints extracts. These effects of glucosamine appear to be critical for the progression of CIOA and result in limited bone remodeling of the joints.

<em>Bone</em> defects in vertebral bodies (VB) usually occur after the reduction of fractures or are caused by <em>bone</em> disease. Besides the treatment of original disease, repair of the <em>bone</em> defect can restore the structure of VB and improve stabilization of the spine to protect the spinal cord nerves. To aid studies of the efficacy of bioengineering techniques for repair of VB, we developed a rat model with a critical size <em>bone</em> defect in VB. Air-motivated burrs were used to create two sizes of <em>bone</em> defect (2 x 3 x 1.5 mm; 2 x 3 x 3 mm) in the anterior part of VB in 6-month-old Fischer 344 rats. Quantitative CT analyses and histological assays demonstrated that neither defects self-repaired by 8 weeks post surgery. Moreover, the tendency of <em>bone</em> formation was monitored in the same animal by serial CT image evaluations, allowing us to demonstrate that there was significant <em>bone</em> growth during the 4- to 6-week period after the creation of the <em>bone</em> defect. We then implanted sintered poly(lactic-co-glycolic acid) (PLGA) microsphere scaffolds loaded with Matrigel with or without recombinant human <em>bone</em> <em>morphogenetic</em> <em>protein</em> 2 (rhBMP2; 2.0 microg rhBMP2/<em>10</em> microL Matrigel/scaffold) into the <em>bone</em> defect (2 x 3 x 3 mm) in the VB. <em>Bone</em> formation was detected by quantitative analyses of serial CT images, which demonstrated <em>bone</em> growth in rats that received the rhBMP2 implant, in both surrounding areas and inside area of the scaffold. In addition to a rapid increase within 2 weeks of the operation, another significant <em>bone</em> formation period was found between 4 and 8 weeks after the implantation. By contrast, the control group that received the implant without rhBMP2 did not show similar <em>bone</em> formation tendencies. The results of CT analyses were confirmed by histological studies. This study suggests that a critical size <em>bone</em> defect of the anterior VB can be developed in a rat model. Characterization of this model demonstrated that 4 to 6 weeks after creation of the defect was a significant <em>bone</em> growth period for VB <em>bone</em> repair in rats. This animal model has further utility for the study of different biomaterials for VB <em>bone</em> repair. Implantation of a bioactive PLGA scaffold carrying rhBMP2 allowed more successful repair of the VB defect. Although further characterization studies are needed, the bioactive PLGA scaffold developed in this study will likely adapt easily to other in vivo osteogenesis applications.