Theresa E. Hefferan

Effect of local sequential VEGF and BMP-2 delivery on ectopic and orthotopic bone regeneration

Bone regeneration is a coordinated cascade of events regulated by several cytokines and growth factors. Angiogenic growth factors are predominantly expressed during the early phases for re-establishment of the vascularity, whereas osteogenic growth factors are continuously expressed during bone formation and remodeling. Since vascular endothelial growth factor (VEGF) and bone morphogenetic proteins (BMPs) are key regulators of angiogenesis and osteogenesis during bone regeneration, the aim of this study was to investigate if their sequential release could enhance BMP-2-induced bone formation. A composite consisting of poly(lactic-co-glycolic acid) microspheres loaded with BMP-2 embedded in a poly(propylene) scaffold surrounded by a gelatin hydrogel loaded with VEGF was used for the sequential release of the growth factors. Empty composites or composites loaded with VEGF and/or BMP-2 were implanted ectopically and orthotopically in Sprague-Dawley rats (n=9). Following implantation, the local release profiles were determined by measuring the activity of (125)I-labeled growth factors using scintillation probes. After 8 weeks blood vessel and bone formation were analyzed using microangiography, microCT and histology. The scaffolds exhibited a large initial burst release of VEGF within the first 3 days and a sustained release of BMP-2 over the full 56-day implantation period. Although VEGF did not induce bone formation, it did increase the formation of the supportive vascular network (p=0.03) in ectopic implants. In combination with local sustained BMP-2 release, VEGF significantly enhanced ectopic bone formation compared to BMP-2 alone (p=0.008). In the orthotopic defects, no effect of VEGF on vascularisation was found, nor was bone formation higher by the combination of growth factors, compared to BMP-2 alone. This study demonstrates that a sequential angiogenic and osteogenic growth factor release may be beneficial for the enhancement of bone regeneration.

Biological Activity of rhBMP-2 Released From PLGA Microspheres

Human recombinant bone morphogenetic protein-2 (rhBMP-2) has been proven effective in stimulating the regeneration of bone in both skeletal and extraskeletal locations. Through encapsulation within, and release from, biodegradable poly(DL-lactic-co-glycolic acid) (PLGA) microspheres, a proven vehicle for sustained delivery of various proteins, the local concentrations of rhBMP-2 could be maintained at optimal levels to stimulate bone regeneration and remodeling at the site of healing in diverse clinical settings. Thus the purpose of this work was to investigate the encapsulation of rhBMP-2 in PLGA microspheres and its biologic activity upon release. Using in vitro tests in simulated body fluids, the effect of rhBMP-2 released from PLGA microspheres upon osteoblast cell cultures was found to be statistically similar to the effect produced by positive controls consisting of nonencapsulated aqueous rhBMP-2 in simulated body fluids. This clarifies an important step in skeletal tissue engineering strategies aimed at the use of encapsulated rhBMP-2 to stimulate bone regeneration and remodeling.

Tissue, cell type, and breast cancer stage‐specific expression of a TGF‐β inducible early transcription factor gene

This laboratory has previously identified a novel TGF‐β inducible early gene (TIEG) in human osteoblasts [Subramaniam et al. (1995): Nucleic Acids Res 23:4907–4912]. Using TIEG specific polyclonal antibody and immunoprecipitation methods in normal human fetal osteoblast cells (hFOB cells), we have now demonstrated that TIEG encodes a 72‐kDa protein whose levels are transiently increased at as early as 2 h of TGF‐β treatment. Polarized confocal microscopic analysis of hFOB cells shows a nuclear localized TIEG protein in untreated cells under the conditions described under Methods. Interestingly, the levels of TIEG protein in the nuclei increase when the cells are treated with TGF‐β1 for 2 h. In contrast, similar analyses of untreated human keratinocytes show a cytoplasmic localized TIEG protein that appears to be translocated to the nucleus after H2O2 treatment. Additional immunohistochemical studies have demonstrated that TIEG protein is expressed in epithelial cells of the placenta, breast, and pancreas, as well as in osteoblast cells of bone and selected other cells of the bone marrow and cerebellum with some cells showing a cytoplasmic localization and others a nuclear localization. All cells of the kidney display negative staining for this protein. Interestingly, a stage specific expression of TIEG protein is found in a dozen breast cancer biopsies, using immunohistochemistry. The cells in normal breast epithelium displays a high expression of TIEG protein, those in the in situ carcinoma display less than one‐half of the levels, and those in the invasive carcinoma show a complete absence of the TIEG protein. TIEG has been localized to chromosome 8q22.2 locus, the same locus as the genes involved in osteopetrosis and acute myeloid leukemia and close to the c‐myc gene locus and a locus of high polymorphism in cancer biopsies. The correlation between the levels of TIEG protein and the stage of breast cancer, its prime location in human chromosome 8q22.2, and past studies with pancreatic carcinoma, suggests that TIEG may play a role in tumor suppressor gene activities, apoptosis, or some other regulatory function of cell cycle regulation. J. Cell. Biochem. 68:226–236, 1998.

COL1 C-propeptide cleavage site mutations cause high bone mass osteogenesis imperfecta

Osteogenesis imperfecta (OI) is most often caused by mutations in the type I procollagen genes (COL1A1/COL1A2). We identified two children with substitutions in the type I procollagen C‐propeptide cleavage site, which disrupt a unique processing step in collagen maturation and define a novel phenotype within OI. The patients have mild OI caused by mutations in COL1A1 (Patient 1: p.Asp1219Asn) or COL1A2 (Patient 2: p.Ala1119Thr), respectively. Patient 1 L1–L4 DXA Z‐score was +3.9 and pQCT vBMD was+3.1; Patient 2 had L1–L4 DXA Z‐score of 0.0 and pQCT vBMD of −1.8. Patient BMD contrasts with radiographic osteopenia and histomorphometry without osteosclerosis. Mutant procollagen processing is impaired in pericellular and in vitro assays. Patient dermal collagen fibrils have irregular borders. Incorporation of pC‐collagen into matrix leads to increased bone mineralization. FTIR imaging confirms elevated mineral/matrix ratios in both patients, along with increased collagen maturation in trabecular bone, compared to normal or OI controls. Bone mineralization density distribution revealed a marked shift toward increased mineralization density for both patients. Patient 1 has areas of higher and lower bone mineralization than controls; Patient 2s bone matrix has a mineral content exceeding even classical OI bone. These patients define a new phenotype of high BMD OI and demonstrate that procollagen C‐propeptide cleavage is crucial to normal bone mineralization. Hum Mutat 32:1–12, 2011.

Estrogen Regulation of a Transforming Growth Factor-β Inducible Early Gene That Inhibits Deoxyribonucleic Acid Synthesis in Human Osteoblasts1

This laboratory reported the identification and characterization of a unique three zinc finger, transcription factor-like, transforming growth factor-beta inducible early gene (TIEG) (see Ref. 35). TIEG expression has been shown to be tissue- and cell type specific, enhanced by specific growth factors, and to decrease with advancing stages of breast cancer. Recent studies involving TIEG overexpression in pancreatic carcinoma cells indicate that TIEG expression inhibits DNA synthesis, similar to a tumor suppressor-like gene, and plays a role in apoptosis (see Ref. 37). This paper describes the rapid, but transient, induction of TIEG steady-state messenger RNA (mRNA) levels by 17beta-estradiol (E2) in estrogen receptor (ER)-positive, human fetal osteoblastic (hFOB/ER) cells. This rapid induction is shown to be ER- and steroid dose-dependent but protein synthesis independent. An antagonism between E2 and PTH, which occurs in skeletal metabolism, is shown to concur rapidly with TIEG mRNA expression. Scanning confocal microscopy (using polarized, laser-based immunofluorescence) shows that TIEG protein is localized in the nucleus of hFOB/ER cells, with the levels rapidly increasing after E2 treatment. The rapid E2-induced increase in TIEG expression is followed by an E2-induced inhibition of DNA synthesis in the hFOB/ER cells. Antiestrogens block not only the induction of TIEG mRNA levels but also the inhibition of cell proliferation. Lastly, hFOB cells, stably transfected with a TIEG expression vector, display markedly reduced DNA synthesis/cell proliferation, compared with nontransfected cells. These results support the finding that TIEG is an early responding regulatory gene for E2 in human osteoblast cells that inhibits DNA synthesis. It is speculated that TIEG may play a role in the signaling pathway for E2 in inhibiting cell proliferation.

Severe Osteogenesis Imperfecta in Cyclophilin B–Deficient Mice

Osteogenesis Imperfecta (OI) is a human syndrome characterized by exquisitely fragile bones due to osteoporosis. The majority of autosomal dominant OI cases result from point or splice site mutations in the type I collagen genes, which are thought to lead to aberrant osteoid within developing bones. OI also occurs in humans with homozygous mutations in Prolyl-3-Hydroxylase-1 (LEPRE1). Although P3H1 is known to hydroxylate a single residue (pro-986) in type I collagen chains, it is unclear how this modification acts to facilitate collagen fibril formation. P3H1 exists in a complex with CRTAP and the peptidyl-prolyl isomerase cyclophilin B (CypB), encoded by the Ppib gene. Mutations in CRTAP cause OI in mice and humans, through an unknown mechanism, while the role of CypB in this complex has been a complete mystery. To study the role of mammalian CypB, we generated mice lacking this protein. Early in life, Ppib-/- mice developed kyphosis and severe osteoporosis. Collagen fibrils in Ppib-/- mice had abnormal morphology, further consistent with an OI phenotype. In vitro studies revealed that in CypB–deficient fibroblasts, procollagen did not localize properly to the golgi. We found that levels of P3H1 were substantially reduced in Ppib-/- cells, while CRTAP was unaffected by loss of CypB. Conversely, knockdown of either P3H1 or CRTAP did not affect cellular levels of CypB, but prevented its interaction with collagen in vitro. Furthermore, knockdown of CRTAP also caused depletion of cellular P3H1. Consistent with these changes, post translational prolyl-3-hydroxylation of type I collagen by P3H1 was essentially absent in CypB–deficient cells and tissues from CypB–knockout mice. These data provide significant new mechanistic insight into the pathophysiology of OI and reveal how the members of the P3H1/CRTAP/CypB complex interact to direct proper formation of collagen and bone.

Effect of Hydrogel Porosity on Marrow Stromal Cell Phenotypic Expression

This study describes investigation of porous photocrosslinked oligo[(polyethylene glycol) fumarate] (OPF) hydrogels as potential matrix for osteoblastic differentiation of marrow stromal cells (MSCs). The porosity and interconnectivity of porous hydrogels were assessed using magnetic resonance microscopy (MRM) as a noninvasive investigative tool that could image the water construct inside the hydrogels at a high-spatial resolution. MSCs were cultured onto the porous hydrogels and cell number was assessed using PicoGreen DNA assay. Our results showed 10% of cells initially attached to the surface of scaffolds. However, cells did not show significant proliferation over a time period of 14 days. MSCs cultured on porous hydrogels had increased alkaline phosphatase activity as well as deposition of calcium, suggesting successful differentiation and maturation to the osteoblastic phenotype. Moreover, continued expression of type I collagen and osteonectin over 14 days confirmed osteoblastic differentiation of MSCs. MRM was also applied to monitor osteogenesis of MSCs on porous hydrogels. MRM images showed porous scaffolds became consolidated with osteogenic progression of cell differentiation. These findings indicate that porous OPF scaffolds enhanced MSC differentiation leading to development of bone-like mineralized tissue.

Collagen type I hydrogel allows migration, proliferation, and osteogenic differentiation of rat bone marrow stromal cells

Hydrogels are potentially useful for many purposes in regenerative medicine including drug and growth factor delivery, as single scaffold for bone repair or as a filler of pores of another biomaterial in which host mesenchymal progenitor cells can migrate in and differentiate into matrix-producing osteoblasts. Collagen type I is of special interest as it is a very important and abundant natural matrix component. The purpose of this study was to investigate whether rat bone marrow stromal cells (rBMSCs) are able to adhere to, to survive, to proliferate and to migrate in collagen type I hydrogels and whether they can adopt an osteoblastic fate. rBMSCs were obtained from rat femora and plated on collagen type I hydrogels. Before harvest by day 7, 14, and 21, hydrogels were fluorescently labeled, cryo-cut and analyzed by fluorescent-based and laser scanning confocal microscopy to determine cell proliferation, migration, and viability. Osteogenic differentiation was determined by alkaline phosphatase activity. Collagen type I hydrogels allowed the attachment of rBMSCs to the hydrogel, their proliferation, and migration towards the inner part of the gel. rBMSCs started to differentiate into osteoblasts as determined by an increase in alkaline phosphatase activity after two weeks in culture. This study therefore suggests that collagen type I hydrogels could be useful for musculoskeletal regenerative therapies.

Non-invasive monitoring of BMP-2 retention and bone formation in composites for bone tissue engineering using SPECT/CT and scintillation probes.

Non-invasive imaging can provide essential information for the optimization of new drug delivery-based bone regeneration strategies to repair damaged or impaired bone tissue. This study investigates the applicability of nuclear medicine and radiological techniques to monitor growth factor retention profiles and subsequent effects on bone formation. Recombinant human bone morphogenetic protein-2 (BMP-2, 6.5 microg/scaffold) was incorporated into a sustained release vehicle consisting of poly(lactic-co-glycolic acid) microspheres embedded in a poly(propylene fumarate) scaffold surrounded by a gelatin hydrogel and implanted subcutaneously and in 5-mm segmental femoral defects in 9 rats for a period of 56 days. To determine the pharmacokinetic profile, BMP-2 was radiolabeled with (125)I and the local retention of (125)I-BMP-2 was measured by single photon emission computed tomography (SPECT), scintillation probes and ex vivo scintillation analysis. Bone formation was monitored by micro-computed tomography (microCT). The scaffolds released BMP-2 in a sustained fashion over the 56-day implantation period. A good correlation between the SPECT and scintillation probe measurements was found and there were no significant differences between the non-invasive and ex-vivo counting method after 8 weeks of follow up. SPECT analysis of the total body and thyroid counts showed a limited accumulation of (125)I within the body. Ectopic bone formation was induced in the scaffolds and the femur defects healed completely. In vivo microCT imaging detected the first signs of bone formation at days 14 and 28 for the orthotopic and ectopic implants, respectively, and provided a detailed profile of the bone formation rate. Overall, this study clearly demonstrates the benefit of applying non-invasive techniques in drug delivery-based bone regeneration strategies by providing detailed and reliable profiles of the growth factor retention and bone formation at different implantation sites in a limited number of animals.

Cytokine-specific induction of the TGF-β inducible early gene (TIEG): Regulation by specific members of the TGF-β family

Select members of the TGF‐β family of cytokines play key regulatory roles in skeletal development, structure, and turnover. This laboratory has previously reported that TGF‐β treatment of immortalized normal human fetal osteoblast (hFOB) cells results in the rapid induction of the mRNA levels of a TGF‐β inducible early gene (TIEG) followed by changes in cell proliferation and bone matrix protein production. Previous studies have also shown that nonmembers of the TGF‐β superfamily showed little or no induction of TIEG mRNA. This article further addresses the cytokine specificity of this TIEG induction by examining whether activin and select bone morphogenetic proteins, (BMP‐2, BMP‐4, and BMP‐6), which are representative of different subfamilies of this superfamily, also induce the expression of TIEG in hFOB cells. However, TGF‐β remained the most potent of these cytokines, inducing TIEG mRNA steady‐state levels at 0.1 ng/ml, with a maximum induction of 24‐fold at 2.0 ng/ml. The BMP‐2 (16‐fold), BMP‐4 (4‐fold), and activin (1–3‐fold) also induced TIEG mRNA levels, but at reduced degrees compared to TGF‐β (24‐fold), and only at much higher cytokine concentrations, e.g., 50–100 ng/ml, compared to 2 ng/ml for TGF‐β. BMP‐6 showed no effect on TIEG mRNA levels. The TIEG protein levels generally correlated with the mRNA steady‐state levels. As with TGF‐β, BMP‐2 treatment of hFOB cells was shown by confocal microscopy to induce a rapid translocation of the TIEG protein to the nucleus. In summary, the relative potencies of these TGF‐β family members to induce TIEG expression generally follows the general osteoinductive capacity of these cytokines, with TGF‐β ⋙ BMP‐2 > BMP‐4 > activin ≫ BMP‐6. J. Cell. Biochem. 78:380–390, 2000.