[Osteoblastic differentiation of human adult mesenchymal stem cells after through gene transfer of BMP-2 in the absence of dexamethasone]. / Osteoblastäre Differenzierung von humanen adulten mesenchymalen Stammzellen durch transgenes BMP-2 in Abwesenheit von Dexamethason.

AIM: Mesenchymal stem cells (MSC) of various species appear to require different cues to differentiate towards the osteoblastic lineage. For MSC of human origin, recombinant hBMP-2 is reported to be not sufficient but dexamethasone seems to be essential. The aim of this study was to analyse changes in genotype and phenotype of hMSC after adenoviral transfer of the BMP-2 gene in the absence of dexamethasone. METHODS: We employed hMSC and analysed changes in expression of the Runx2, Osterix and type I collagen gene by quantitative PCR after adenoviral transfer of the human BMP-2 gene in the absence of dexamethasone. As a phenotypic marker alkaline phosphatase activity was assessed. ANOVA and post hoc statistical analyses were used to determine differences among data (p < 0.05). RESULTS: Transfer of the hBMP-2 gene and consecutive production of transgenic BMP-2 up-regulated bone marker gene expression and increased alkaline phosphatase activity and thus promoted an enhanced lineage progression to the osteoblast phenotype without the addition of dexamethasone. CONCLUSION: These findings are noteworthy in the light of a possible superiority of endogenous transgenic proteins compared to exogenous recombinant proteins.

[Refined adenoviral transduction for controlled gene transfer into human adult mesenchymal stem cells]. / Kontrollierter Gentransfer in humane adulte mesenchymale Stammzellen mittels adenoviraler Vektoren.

AIM: Adenoviral gene transfer remains a powerful tool for basic research purposes. We hypothesize that adenoviral transduction of human mesenchymal stem cells (hMSC) in vitro can be improved by refined use of experimental parameters. METHODS: hMSCs were transduced by adenoviral vectors encoding Luciferase or BMP-2 at a selection of multiplicities of infection (MOI) and exposure times. Transgene production and total protein content were measured. To determine practical relevance, expression of the bone marker genes Runx2 and Type I collagen was analyzed by quantitative PCR. As a phenotypic marker alkaline phosphatase was assessed. ANOVA and post hoc statistical analyses were used to determine differences among data (p < 0.05). RESULTS: Prolonged exposure led to a decrease in transgene production and total protein content. Increasing MOI at exposure of up to 4 hours resulted in a higher production of the transgene. Transfer of the hBMP-2 gene promoted an enhanced lineage progression to the osteoblast phenotype indicating biological activity. CONCLUSION: Time of exposure is of major importance for toxicity in vitro and should not exceed 4 hours for hMSC. While increase in exposure time leads to cell death, surviving cells, up to a certain limit, seem to compensate by increasing production of the transgene indicating that transduction efficiency cannot be positively measured in a binary yes-or-no scheme.

[The effect of growth/differentiation factor-5 (GDF-5) on genotype and phenotype in human adult mesenchymal stem cells]. / Effekt von Growth/Differentiation Factor-5 (GDF-5) auf Genotyp und Phänotyp humaner adulter mesenchymaler Stammzellen.

AIM: To evaluate the effects of GDF-5 on genotype and phenotype of human mesenchymal stem cells (hMSC). HYPOTHESIS: GDF-5 leads to up-regulation of the Type I-collagen (Col) gene without altering bone marker genes or alkaline phosphatase activity. METHODS: To test our hypothesis hMSC were treated with rmGDF-5. Using quantitative real-time PCR we analyzed mRNA for Col, Runx2 and Osterix (Osx). Furthermore, we analyzed alkaline phosphatase activity (ALP) as a phenotypical bone marker. ANOVA and post hoc statistical analyses were used to determine differences among treatments (p < 0.05). RESULTS: HMSC showed a biphasic response in both Col and Runx2 after rmGDF-5. Initial up-regulation was followed by a significant down-regulation below controls. Interestingly, the controls presented with changes for Col and Runx2 over time. There was no Osx expression in either treated hMSC or controls. No significant differences could be detected in ALP. CONCLUSION: Increased expression of Col and Runx2 might indicate differentiation towards both osteoblast and fibroblast lineage. However, no Osx expression and no change in ALP support the assumption that rmGDF-5 does not lead to an osteoblast phenotype in hMSC. Our in vitro studies confirm a possible therapeutic benefit of GDF-5 in the treatment of tendon and ligament injuries and tissue engineering approaches. Further research is necessary to prove its clinical value.

Rabbit calvarial wound healing by means of seeded Caprotite scaffolds.

Autologous bone is the most successful bone-grafting material; however, limited supply and donor site morbidity are problematic. Synthetic bone substitutes are effective, but healing is slow and unpredictable. Osseous wound healing may be enhanced if bone substitutes are combined with autologous bone marrow cells. To test this hypothesis, we created 40 calvarial defects in 20 12-week-old New Zealand White rabbits, divided into four groups: (1) unrepaired controls, (2) autologous bone grafts, (3) unseeded Caprotite (a polymer-ceramic composite) grafts, and (4) Caprotite grafts seeded with autologous bone marrow stromal cells. CT scans were obtained at 0, 6, and 12 weeks post-operatively, and defects were harvested for histology. Defects repaired with autologous bone had significantly (p < 0.05) more bone than the other three groups, although seeded Caprotite defects showed different wound-healing sequelae. Results suggest that seeded Caprotite scaffolds did not significantly enhance osseous defect healing compared with controls.

Controlled release from coated polymer microparticles embedded in tissue-engineered scaffolds.

The controlled release of proteins in tissue-engineered implants is being examined with the potential application to improve vascularization and hasten tissue growth. Bovine serum albumin (BSA), was encapsulated within poly(D,L-lactic-co-glycolic acid) [PLGA] microparticles. The microparticles were coated with poly(vinyl alcohol) and incorporated into PLGA tissue-engineered scaffolds during fabrication. The release of BSA from PLGA microparticles, coated PLGA microparticles, and microparticles embedded in a porous PLGA scaffold was measured. We have developed a novel approach that will permit incorporation of coated polymeric microparticles during PLGA scaffold fabrication. Growth factors or drugs could be incorporated into the microparticles resulting in a long-term, controlled release.

Therapeutic potential of bone morphogenetic proteins.

Recently, there has been substantial progress in the area of bone morphogenetic protein (BMP) research. This review serves as an up-to-date summary of the history of BMPs, the mechanisms of BMP signalling and the role of BMPs in adipose, kidney, liver, bone and nervous system. The potential of BMPs as therapeutic agents will also be discussed.

An osteogenic cell culture system to evaluate the cytocompatibility of Osteoset, a calcium sulfate bone void filler.

The purpose of the study was to describe a convenient, reliable and quantitative in vitro assay system to assess the cytocompatibility of a calcium sulfate bone filler on two osteogenic cell lines and primary osteoblasts. The hypothesis was that the bone void filler, OsteoSet pellets, would not impact adversely on cell proliferation kinetics or osteogenic potential of selected cells. The hypothesis was tested by standard in vitro methodology of placing OsteoSet pellets either directly in contact with osteogenic cells, or by compartmentalizing within transwell - clear microporous membrane inserts. Data analyses were accomplished with appropriate post hoc statistics (p < or = 0.05). In the presence of the OsteoSet pellets, the cell lines exhibited a decrease in cell proliferation at days 4 and 7, independent of either cell type or tissue culture medium. A decrease in the alkaline phosphatase enzyme activity occurred in the osteogenic cell lines maintained for 9 and 16 days in the presence of the OsteoSet pellets. However, with the exception of the MC3T3E-1 line, no differences were observed with respect to calcium deposition (mineralization) by day 16. Intact human osteocalcin release data for the human-derived OPC1 line and the primary osteoblasts was inconclusive as the OsteoSet pellets may interact with the osteocalcin secreted into the tissue culture medium. The present studies describe a cell culture system to assess the cytocompatibility of bone-graft substitutes with osteogenic cells by compartmentalizing material from direct cell contact (in transwells), and additionally, by evaluating direct cell/biomaterial interactions.

Options for tissue engineering to address challenges of the aging skeleton.

There will be more than 52 million Americans over the age of 65 by the year 2020 (U.S. Census Bureau). Regenerating form and function to bone defects in an elderly, osteoporotic population of this magnitude will be a daunting challenge. Tissue engineering options must be considered to answer this challenge. Options can include gene transfer technology, stem cell therapy, and recombinant signaling molecules. An additional component will be a carrier that localizes, protects, predictably releases cues and cells, as well as establishes an environment for restoring osseous form and function. The purposes of this article are to present an overview of the bone regenerating decrement affecting osteoporotic, elderly patients and to highlight some tissue engineering options that could offset this decrement.

Gene therapy approaches for modulating bone regeneration.

Following injury, bone has the ability to regenerate itself to a form and function nearly indistinguishable from the pre-injury state. However, if the injury is beyond a critical limit, recovery will not occur without therapeutic interventions. Autografts and implants with banked bone continue as the treatments of choice, although each exhibits limitations and liabilities. Alternatives have included the utilization of bone-graft substitutes that may incorporate bone derivatives and soluble signaling molecules such as mitogens and morphogens. In addition, an evolving treatment modality, gene therapy, offers an exciting avenue for bone regeneration. This review presents some of the current concepts for developing a rational gene therapy approach in bone regeneration.

Nicotine administration in rabbits using Habitrol nicotine patches and nicotine nasal spray.

1. Several methods are used to provide predictable and effective nicotine to experimental animals in scientific studies. Due to the expense and technical challenges of these methods, we sought suitable alternatives. Consequently, the purpose of the present study was to develop a reliable experimental nicotine protocol in rabbits that included either Habitrol nicotine patches (Novartis Consumer Health Inc., Summit, NJ, USA) or nicotine nasal spray. 2. Administration of one of three doses of nicotine (2.5, 5, or 10 mg) was accomplished daily on 13 rabbits divided between either the patch or spray groups. Systemic nicotine and cotinine levels at 0 h, 15 min and 8 and 24 h were assayed. Data were analysed by a Fisher's protected least significant difference test at P = 0.05. 3. Rabbits treated with Habitrol patches exhibited consistent and predictable systemic nicotine levels. The nicotine nasal spray produced an immediate dose-dependent response with no measurable nicotine serum levels at 8 h. 4. For nicotine administration in rabbits, nicotine patches are easy to administer and provide a nicotine serum level between 5 and 25 ng/mL, which is consistent with the average daily level found in a patient who smokes cigarettes.

Histomorphometry of parietal bones versus age and race.

Surgeons are used to obtaining bone grafts of calvaria, which are abundant and available. The outer table of the parietal bone can be split, usually at the level of the diploic interstice, with an osteotome. Inadvertently violating the inner table and the dura leads some surgeons to avoid using the outer table in the elderly. Sullivan and Smith measured the thickness of the outer tables, diploe, and inner table of 37 cadavers (average age, 59 years) and found each layer to be well preserved distinctly. However, they suggested that calvaria become brittle in patients older than 50 years of age. The current authors hypothesized that diploic composition is not changed, even in the elderly. The thickest part of the parietal bone of 49 Koreans and 30 whites were acquired, and undecalcified slides were made. Via light microscopy, using the National Institutes of Health image, the following measurements were made: the thickness of the parietal bone (PT), outer table (OT), diploe (DT), and inner table (IT); trabecular bone volume percent (TBV); trabecular thickness (TT); and trabecular separation (TS). There was no significant difference in the thickness of the OT, DT, and IT of the PT, TBV, TT, and TS among different ages. The PTs of women were thicker than men's. The PTs of whites were significantly thicker than Koreans'. This study disclosed that the DT is not different among varying age groups and is not sclerosed in the persons older than 80 years, and the OT of these individuals is not brittle. Thus, surgeons do not need to hesitate when taking the OT of the PT in older patients. However, the selection of the site is attentive to PT. The thickest posteromedial part of the PT is favorable and safe as a donor.

Interface between titanium miniplate/screw and human calvaria.

This study demonstrates interfacial changes of titanium miniplate/screw with normal calvaria and nonvascularized calvarial bone graft ensued from craniectomy in a 53-year-old female. In 18 months after operation, a right parietal bone containing an L-shaped miniplate with screws, 5 mm long and 2 mm in external diameter, was harvested, fixed, and embedded in methylmetacrylate. Fifteen micrometer thick sections were made by an EXAKT cutting-grinding system (Exakt Company, Hamburg, Germany), and reviewed under the bright field light microscope. The mean of the bone-contacting surface ratio (BCSR) in six screwed bone was 64.1%; 69.3% in normal bone, and 60.4% in grafted bone. The trabecular bone areas in 10 x 5 mm (50 mm2) rectangular area of diploe surrounding the screws was 43.02 mm2; 45.25 mm2 in normal calvaria; and 41.82 mm2 in grafted bone. The mean Ca/P peak-height ratio of the plated and screwed calvaria was 1.47 in a 9 mm wide zone around each screw; 1.37 in normal calvaria; and 1.51 in grafted calvaria. We concede that the effect of direct contact of titanium with screws onto the bone is as much as an osseous integration (osseointegration).

Carrier systems for bone morphogenetic proteins.

Bone deficits can regenerate inherently, although when the amount of bone loss exceeds a critical limit, pseudarthrosis and fibrosis occur. Therapeutic intervention either with an autograft or allogeneic bank bone are traditional options to promote regeneration to overcome critical limits. However, liabilities with traditional treatments have inspired investigators to develop alternatives, such as combinations of biomimetic scaffolds and osteogenic regulatory molecules. The class of osteogenic regulatory molecules known as the bone morphogenetic proteins has several members that stimulate bone regeneration. Therapeutic applications of bone morphogenetic proteins require a well characterized carrier system to ensure safe and effective presentation at the implant site. Several carrier systems have been used to evaluate the sustained release and implant retention of recombinant human bone morphogenetic protein-2. The carrier systems used in this study include type I collagen, poly(D,L-lactide), and deorganified bovine bone. Pharmacokinetics of recombinant human bone morphogenetic protein-2 released from these systems were characterized in the rat ectopic assay. Pharmacokinetics were influenced by the implant carrier. For example, sustained release occurred with the collagen sponge. The recombinant human bone morphogenetic protein-2 from deorganified bovine bone resulted in a burst release at the first collection interval, but thereafter, appeared to bind irreversibly to the morphogen. The poly (D,L-lactide) systems showed a dose dependent sustained release pattern. These results indicate the physicochemical characteristics of a carrier system for recombinant human bone morphogenetic protein-2 impact the release kinetics and may have a profound influence on clinical outcome.

Reconstruction of bone using calcium phosphate bone cements: a critical review.

The calcium phosphate cements (CPCs) are rapidly emerging as a new technology in craniofacial surgery and will soon impact many areas of orthopedic and maxillofacial reconstructive surgery as well. These materials are, in many ways, substantially different from the previously marketed dense, crystalline, hydroxyapatite (HA) ceramic materials of the 1980s. The CPCs are blends of amorphous and crystalline calcium phosphate compounds and set to produce HA. These materials 1) have x-ray diffraction spectra similar to the mineral phase of bone, 2) set endothermically at body temperature, 3) are capable of being injected into fractures or bone defects, 4) have compressive strengths equal to or greater than bone, 5) form chemical bonds to the host bone, and 6) may exhibit osteoconductive properties. This review provides an overall commentary on the different types of CPCs, emphasizing those materials currently on the market or soon to emerge in the marketplace.

Establishing an immortalized human osteoprecursor cell line: OPC1.

The present studies evaluated the feasibility of establishing a conditionally immortalized osteoprecursor cell line derived from human fetal bone tissue. Primary cultures were transfected with a plasmid in which the Mx-1 promoter drives the expression of SV40 T-antigen when activated by human A/D interferon. Several neomycin (G418)-resistant colonies were characterized for cell growth and alkaline phosphatase (ALP) enzyme activity. The clone, designated OPC1 (osteoblastic precursor cell line 1), which exhibited the highest ALP enzyme activity at passage 10 (P10), was selected for additional osteogenic phenotypic characterization. Reverse transcription-polymerase chain reaction (RT-PCR) phenotyping revealed abundant mRNA for osteocalcin (OC), osteonectin (ON), osteopontin (OP), parathyroid hormone receptor (PTHr), ALP, and procollagen type I (ProI). In addition, the levels of quantitative RT-PCR product of ON, OP, PTHr, and ProI mRNAs exhibited a marked up-regulation when maintained in medium containing an osteogenic supplement (OS). The ability to stimulate osteogenic differentiation was characterized in postconfluent OPC1 cells maintained in tissue culture medium supplemented with recombinant human bone morphogenetic protein-2 (rhBMP-2) either with or without an OS. All treatment groups exhibited a striking up-regulation of ALP enzyme activity that coincided with ALP histochemical observations. Postconfluent cells also exhibited the ability to form mineralized nodules under all treatments (confirmed by von Kossa histochemical staining and calcium deposition). An enzyme immunosorbent assay (EIA) was utilized to measure intact human OC from the OPC1 line under the various treatments. Abundant OC was evident in the tissue culture medium indicating de novo sythesis and release from the OPC1 line under appropriate conditions. The clonal human-derived OPC1 line represents a homogeneous osteogenic cell line that not only has maintained a consistent bone phenotype from P10 to at least P30, but has also exhibited the capacity to generate programmed differentiation in the presence of low dose rhBMP-2 (10 ng/ml). Thus, the OPC1 line is a human-derived osteoprecursor that provides a sensitive in vitro cell culture system to evaluate bone development, cell/biomaterial interactions, and may be a useful screen for putative bone differentiating factors.

Tissue engineering of bone in the craniofacial complex.

Tissue engineered therapies to regenerate bone in the craniofacial complex will probably include combinations of BMP-like molecules, a BMP-responsive set of cells (both endogenous and exogenous), and packaging in a surgically convenient format. In this report we have described our work with OPCs, BMP, and polymer: components suitable for tissue engineering.

Eighteen-month radiographic and histologic evaluation of sinus grafting with anorganic bovine bone in the chimpanzee.

Maxillary sinus grafting procedures are currently the treatment of choice when the alveolar crest of the posterior maxilla is in close approximation to the maxillary sinus. The short-term histologic and radiographic healing following sinus grafting with natural bone mineral (Bio-Oss) in the chimpanzee has been evaluated. We have previously shown by histomorphometric and radiographic analysis that the percentage of vital bone area, the vertical height, and the density of new bone in the maxillary sinus was significantly greater with anorganic bovine bone compared to bovine Type I collagen matrix. The purpose of this in vivo study was to determine the bone mineral density (BMD) of the sinus grafts, the vertical height stability, the vital bone area, and the extent of anorganic bovine bone replacement 18 months postoperatively in 4 maxillary sinuses from 4 different animals. Radiographic analysis of computed tomographic scans taken at 1.5 years revealed an average BMD of 658 mg/mL, which was not significantly different from the values found at 6.5 months. The radiographic vertical height was maintained between the 6.5- and 18-month time points. On average, the grafts were found to have a height of 14 mm. Lateral wall biopsy specimens at 7.5 months were compared to those at 18 months. With the anorganic bovine bone treatment, the percentage of vital bone area increased from 62 +/- 3% to 70 +/- 7% and the percentage of natural bone mineral area decreased from 19 +/- 14% to 6 +/- 3%. The bovine Type I collagen matrix vital bone percentage at 7.5 months was 34 +/- 21%. These results demonstrate that sinus grafting with anorganic bovine bone maintains radiographic evidence of density and height stability of 1.5 years. In addition, histologic evidence supports the hypothesis that anorganic bovine bone is replaced by vital bone.

Impact of nicotine on bone healing.

A limited number of experimental animal studies and in vitro data confirm that nicotine impairs bone healing, diminishes osteoblast function, causes autogenous bone graft morbidity, and decreases graft biomechanical properties. Therefore, our long-term goal is to develop an effective therapy to reverse the adverse impact of nicotine from tobacco products. However, before accomplishing this goal, we had to develop an animal model. Our hypotheses were nicotine administration preceding and following autogenous bone grafting adversely affected autograft incorporation and depressed donor site healing in a characterized animal wound model. Hypothesis testing was accomplished in bilateral, 4-mm diameter parietal bone defects prepared in 60 Long-Evans rats (male, 35-day-old). A 4-mm diameter disk of donor bone was removed from the left parietal bone and placed in the contralateral defect. The donor site served as a spontaneously healing bone wound. The rats were partitioned equally among three doses of nicotine administered orally in the drinking water (12.5, 25, and 50 mg/L). For each dose, the duration and sequence of nicotine treatment followed four courses, including no nicotine and designated combinations of nicotine administration and abatement prior to and following osseous surgery. Experimental sites were recovered on 14 and 28 days postsurgery, responses quantitated, and data analyzed by analysis of variance and post hoc statistics (p < or = 0.05). We developed a convenient and effective osseous model, and the results validated our hypothesis that nicotine negatively impacts on bone healing.