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P H Krebsbach

Publications and source records attributed to P H Krebsbach.

At least 19 recordsLinked to original sources
PubMed2006

Transplanted endothelial cells enhance orthotopic bone regeneration.

The aim of this study was to determine if endothelial cells could enhance bone marrow stromal-cell-mediated bone regeneration in an osseous defect. Using poly-lactide-co-glycolide scaffolds as cell carriers, we transplanted bone marrow stromal cells alone or with endothelial cells into 8.5-mm calvarial defects created in nude rats. Histological analyses of blood vessel and bone formation were performed, and microcomputed tomography (muCT) was used to assess mineralized bone matrix. Though the magnitude of the angiogenic response between groups was the same, muCT analysis revealed earlier mineralization of bone in the co-transplantation condition. Ultimately, there was a significant increase (40%) in bone formation in the co-transplantation group (33 +/- 2%), compared with the transplantation of bone marrow stromal cells alone (23 +/- 3%). Analysis of these data demonstrates that, in an orthotopic site, transplanted endothelial cells can influence the bone-regenerative capacity of bone marrow stromal cells.

Absorbable Implants↗
PubMed2006

Craniofacial tissue engineering by stem cells.

Craniofacial tissue engineering promises the regeneration or de novo formation of dental, oral, and craniofacial structures lost to congenital anomalies, trauma, and diseases. Virtually all craniofacial structures are derivatives of mesenchymal cells. Mesenchymal stem cells are the offspring of mesenchymal cells following asymmetrical division, and reside in various craniofacial structures in the adult. Cells with characteristics of adult stem cells have been isolated from the dental pulp, the deciduous tooth, and the periodontium. Several craniofacial structures--such as the mandibular condyle, calvarial bone, cranial suture, and subcutaneous adipose tissue--have been engineered from mesenchymal stem cells, growth factor, and/or gene therapy approaches. As a departure from the reliance of current clinical practice on durable materials such as amalgam, composites, and metallic alloys, biological therapies utilize mesenchymal stem cells, delivered or internally recruited, to generate craniofacial structures in temporary scaffolding biomaterials. Craniofacial tissue engineering is likely to be realized in the foreseeable future, and represents an opportunity that dentistry cannot afford to miss.

Absorbable Implants↗
PubMed2005

Engineering craniofacial scaffolds.

OBJECTIVE: To develop an integrated approach for engineering craniofacial scaffolds and to demonstrate that these engineered scaffolds would have mechanical properties in the range of craniofacial tissue and support bone regeneration for craniofacial reconstruction. EXPERIMENTAL VARIABLE: Scaffold architecture designed to achieve desired elasticity and permeability. Scaffold external shape designed to match craniofacial anatomy. OUTCOME MEASURE: Final fabricated biomaterial scaffolds. Compressive mechanical modulus and strength. Bone regeneration as measured by micro-CT scanning, mechanical testing and histology. SETTING: Departments of Biomedical Engineering, Oral/Maxillofacial Surgery, and Oral Medicine, Pathology and Oncology at the University of Michigan. RESULTS: Results showed that the design/fabrication approach could create scaffolds with designed porous architecture to match craniofacial anatomy. These scaffolds could be fabricated from a wide range of biomaterials, including titanium, degradable polymers, and degradable calcium phosphate ceramics. Mechanical tests showed that fabricated scaffolds had compressive modulus ranging 50 to 2900 MPa and compressive strength ranging from 2 to over 56 MPa, within the range of human craniofacial trabecular bone. In vivo testing of designed scaffolds showed that they could support bone regeneration via delivery of BMP-7 transduced human gingival fibroblasts in a mouse model. Designed hydroxyapatite scaffolds with pore diameters ranging from 400 to 1200 microns were implanted in minipig mandibular defects for 6 and 18 weeks. Results showed substantial bone ingrowth (between 40 and 50% at 6 weeks, between 70 and 80% at 18 weeks) for all scaffolds, with no significant difference based on pore diameter. CONCLUSION: Integrated image-based design and solid free-form fabrication can create scaffolds that attain desired elasticity and permeability while fitting any 3D craniofacial defect. The scaffolds could be manufactured from degradable polymers, calcium phosphate ceramics and titanium. The designed scaffolds supported significant bone regeneration for all pore sizes ranging from 300 to 1200 microns. These results suggest that designed scaffolds are clinically applicable for complex craniofacial reconstruction.

Animals↗
PubMed2005

Tissue engineering osteochondral implants for temporomandibular joint repair.

Tissue engineering has provided an alternative to traditional strategies to repair and regenerate temporomandibular joints (TMJ). A successful strategy to engineer osteochondral tissue, such as that found in the TMJ, will produce tissue that is both biologically and mechanically functional. Image-based design (IBD) and solid free-form (SFF) fabrication can be used to generate scaffolds that are load bearing and match patient and defect site geometry. The objective of this study was to demonstrate how scaffold design, materials, and biological factors can be used in an integrated approach to regenerate a multi-tissue interface. IBD and SFF were first used to create biomimetic scaffolds with appropriate bulk geometry and microarchitecture. Biphasic composite scaffolds were then manufactured with the same techniques and used to simultaneously generate bone and cartilage in discrete regions and provide for the development of a stable interface between cartilage and subchondral bone. Poly-l-lactic acid/hydroxyapatite composite scaffolds were differentially seeded with fibroblasts transduced with an adenovirus expressing bone morphogenetic protein-7 in the ceramic phase and fully differentiated chondrocytes in the polymeric phase, and were subcutaneously implanted into mice. Following implantation in the ectopic site, the biphasic scaffolds promoted the simultaneous growth of bone, cartilage, and a mineralized interface tissue. Within the ceramic phase, the pockets of tissue generated included blood vessels, marrow stroma, and adipose tissue. This combination of IBD and SFF-fabricated biphasic scaffolds with gene and cell therapy is a promising approach to regenerate osteochondral defects and, ultimately, the TMJ.

Animals↗
PubMed2003

Indirect solid free form fabrication of local and global porous, biomimetic and composite 3D polymer-ceramic scaffolds.

Precise control over scaffold material, porosity, and internal pore architecture is essential for tissue engineering. By coupling solid free form (SFF) manufacturing with conventional sponge scaffold fabrication procedures, we have developed methods for casting scaffolds that contain designed and controlled locally porous and globally porous internal architectures. These methods are compatible with numerous bioresorbable and non-resorbable polymers, ceramics, and biologic materials. Phase separation, emulsion-solvent diffusion, and porogen leaching were used to create poly(L)lactide (PLA) scaffolds containing both computationally designed global pores (500, 600, or 800 microm wide channels) and solvent fashioned local pores (50-100 microm wide voids or 5-10 microm length plates). Globally porous PLA and polyglycolide/PLA discrete composites were made using melt processing. Biphasic scaffolds with mechanically interdigitated PLA and sintered hydroxyapatite regions were fabricated with 500 and 600 microm wide global pores. PLA scaffolds with complex internal architectures that mimicked human trabecular bone were produced. Our indirect fabrication using casting in SFF molds provided enhanced control over scaffold shape, material, porosity and pore architecture, including size, geometry, orientation, branching, and interconnectivity. These scaffolds that contain concurrent local and global pores, discrete material regions, and biomimetic internal architectures may prove valuable for multi-tissue and structural tissue interface engineering.

Biomechanical Phenomena↗
PubMed2001

Role of Cbfa1 in ameloblastin gene transcription.

Ameloblastin is a tooth-specific extracellular matrix protein that is thought to play a role in enamel crystal formation in the developing dentition. The murine ameloblastin promoter functions in a cell type-specific manner and contains cis-acting elements that function both to enhance and to suppress transcription. The objective of this study was to determine whether the transcription factor Cbfa1, known to be essential for transcription of other mineralized tissue genes, is also required for ameloblastin transcription. Site-directed mutagenesis of the Cbfa1-binding site (-248 base pairs) termed osteoblast-specific element 2 (OSE2) decreased ameloblastin promoter activity by greater than 50% in ameloblast-like cells. No differences in promoter activity were observed in two other oral tissue-derived cell lines transfected with similar constructs. Nuclear factor binding to the ameloblastin promoter was also shown to be cell type-specific and was altered by site-specific mutations in the OSE2 site. Cbfa1 was specifically shown to participate in the DNA-protein complexes between nuclear factors and the ameloblastin OSE2 site by supershift electrophoretic mobility shift assays. The findings that Cbfa1 interacts with functionally important regions of the ameloblastin promoter while promoter activity is diminished in constructs containing site-directed mutations in the Cbfa1 site indicate that Cbfa1 possesses an important function in transcription of the ameloblastin gene.

Animals↗
PubMed2001

Pedicled bone flap formation using transplanted bone marrow stromal cells.

HYPOTHESIS: Transplanted osteoprogenitor cells derived from cultured bone marrow stromal cells (BMSCs) can be used to fabricate pedicled bone flaps. DESIGN: Prospective, randomized experimental trials. SETTING: Basic science research laboratory. MATERIALS: Immunodeficient female NIH-Bg-Nu-Xid mice, aged 3 months. INTERVENTION: The BMSCs were harvested from the long bones of C57Bl/6 transgenic mice carrying the type Ialpha1 collagen-chloramphenicol acetyl transferase reporter gene construct; their numbers were expanded in tissue culture. Treated mice received BMSC transplantations around the common carotid artery and internal jugular vein, the aorta and its venae comitantes, or the saphenous artery and vein; control mice received a sham transplant in comparable recipient sites. MAIN OUTCOME MEASURES: Mice underwent harvesting from 4 weeks to 2 years after transplantation. Transplants were evaluated via histological, immunohistochemical, and angiographic analyses. RESULTS: Compared with the controls, which formed no bone, 32 of 37 BMSC-containing transplants formed a vascularized bone island that was perfused specifically and solely by its common carotid artery vascular source. Mature transplants consisted of well-developed lamellar, corticocancellous bone whose osteocytes were derived from the grafted BMSCs; hematopoietic tissue derived from the recipient mouse. Transplants formed as early as 4 weeks and remained stable in size as late as 108 weeks. CONCLUSIONS: Bone marrow stromal cells can be used to create vascularized bone flaps in mice; these bone constructs are vascularized by their pedicle and therefore can potentially be transferred to a recipient site using microsurgical techniques. These findings provide proof of principle of an additional clinical application of BMSC transplantation techniques.

Animals↗
PubMed2001

Image-based biomimetic approach to reconstruction of the temporomandibular joint.

This article will present an image-based approach to the designing and manufacturing of biomimetic tissue engineered temporomandibular (TMJ) condylar prosthesis. Our vision of a tissue-engineered TMJ prosthesis utilizes a 3-D designed and manufactured biodegradable scaffold shaped similar to a condylar head and neck, i.e. a condylar-ramus unit (CRU). The fabricated CRU scaffold can be constructed with a specific intra-architectural design such that it will enhance the formation of tissue from implanted cells placed within its interstices. These biologic cues could influence scaffold-implanted mesenchymal stem cells (MSC) or bone marrow stromal cells (BMSC) to form a fibrocartilaginous joint surface, or cap, on top of a bony strut, similar to a costochondral rib graft (CCRG), which could be fixed to the mandibular ramus. This new approach to tissue engineering a TMJ would be advantageous because of its patient site-specific anatomical configuration as well as its potential ability to adapt to the loading forces placed on it during function.

Biocompatible Materials↗
PubMed2000

Gene therapy for bone formation: in vitro and in vivo osteogenic activity of an adenovirus expressing BMP7.

Bone morphogenetic proteins (BMPs) are well-established agents for inducing orthotopic and ectopic bone formation. However, their clinical usefulness as regenerative agents may be limited by a short in vivo half-life and low specific activity. BMP gene therapy is an alternative route for exploiting the bone-inductive activity of this class of molecules. To test the feasibility of this approach, we examined the osteogenic activity of AdCMV-BMP7, an adenovirus containing BMP7 cDNA under control of the CMV promoter that was constructed using Cre/lox recombination (Hardy et al. [1997] J. Virol. 71:1842-1849). Adenovirus vectors were shown to readily infect a wide variety of cell types in vitro including osteoblasts, fibroblasts, and myoblasts. COS7 cells transduced with AdCMV-BMP7 produced high levels of BMP-7 (approximately 0.5 microg/10(6) cells). Furthermore, transduction of C2C12 murine myoblast cells with AdCMVBMP-7 suppressed the muscle phenotype and induced in vitro osteoblast differentiation. To test its in vivo biological activity, AdCMV-BMP7 was mixed with a bovine bone-derived collagen carrier (10(8) plaque-forming units virus/site) and was implanted into mouse muscle and dermal pouches. In both cases, an ossicle containing cortical and trabecular bone and a clearly defined marrow cavity formed at the site of virus implantation within 4 weeks. These data demonstrate that AdCMV-BMP7 transduced cells produce biologically active BMP-7 both in vitro and in vivo and show that gene therapy by direct viral transduction using a virus/matrix implant may be a viable route for stimulating bone regeneration.

Adenoviridae↗
PubMed2000

Gene therapy-directed osteogenesis: BMP-7-transduced human fibroblasts form bone in vivo.

An ex vivo gene therapy strategy was used to achieve localized skeletal regeneration in vivo. When an adenovirus vector engineered to express bone morphogenetic protein 7 transduced human gingival fibroblasts or rat dermal fibroblasts, these nonosteogenic tissues formed bone and supported the development of hematopoietic tissue when transplanted into immunocompromised mice. Transduced gingival fibroblasts formed marrow-containing ossicles in 100% of transplants after 1-2 weeks in vivo (n = 30). Immunostaining with murine and human-specific antisera raised against osteonectin and in situ hybridization of human-specific Alu genomic sequence demonstrated that the newly formed bone organ was a chimera of both the human donor and the mouse recipient cells. In experiments of greater clinical relevance, AdCMVBMP-7-transduced dermal fibroblasts repaired critical size skeletal defects in rat calvariae (n = 6). The results of this study suggest a bifunctional role of BMP-7-transduced fibroblasts. The transduced, nonosteogenic cells not only secreted biologically active BMP-7 in vitro and in vivo, but also differentiated into bone-forming cells in vivo. This model exploits the use of an easily biopsied, self-regenerating tissue such as gingiva or skin and suggests that local regeneration of tissues by ex vivo gene therapy may not require that autogenous cells be cultured from the tissue that is to be regenerated.

Adenoviridae↗
PubMed2000

Cloning, characterization and immunolocalization of human ameloblastin.

Amelogenesis imperfecta is a broad classification of hereditary enamel defects, exhibiting both genetic and clinical diversity. Most amelogenesis imperfecta cases are autosomal dominant disorders, yet only the local hypoplastic form has been mapped to human chromosome 4q between D4S242 1 and the albumin gene. An enamel protein cDNA, termed ameloblastin (also known as amelin and sheathlin), has been isolated from rat, mouse and pig. Its human homolog has been mapped to chromosome 4q21 between markers D4S409 and D4S400, flanking the local hypoplastic amelogenesis imperfecta critical region. Therefore, ameloblastin is a strong candidate gene for this form of amelogenesis imperfecta. To facilitate genetic studies related to this dental disease, we isolated and characterized a human ameloblastin cDNA. A human third molar cDNA library was screened and two ameloblastin clones identified. Nucleotide sequencing of these cDNAs indicated alternative splicing of the putative open reading frame, use of different polyadenylation signals, and a high degree of similarity to reported rat, mouse and porcine cDNAs. Immunohistochemistry studies on embryonic human teeth using an antibody to recombinant ameloblastin indicated ameloblastin expression by ameloblasts with localization in the enamel matrix associated with the sheath structures.

Alternative Splicing↗
PubMed2000

[A tissue engineering approach to site-specific reconstruction of skeletal structures of the maxillofacial region:part I]

This article will present an image-based approach to design and manufacture of scaffolds for the tissue engineering of bone and cartilage constructs. Our vision of tissue-engineered bone and/or cartilage constructs utilizes 3-D designed and manufactured biodegradable scaffolds that are site specific for the area of maxillofacial reconstruction. The fabricated site-specific scaffold can be constructed such that it will impart biologic cues to implanted cells placed within its interstices. These biologic cues should influence scaffold implanted mesenchymal stem cells (MSC) or bone marrow stromal cells (BMSC) to form the necessary tissue for site specific facial reconstruction. This new approach to tissue engineering of the maxillofacial region would be advantageous because of its site-specific anatomical configuration as well as its potential ability to adapt to the functional forces placed on it during function. The reconstruction of the condylar-ramus area of the temporomandibular joint (TMJ) will be used as an illustration of this approach.

Journal Article↗
PubMed1999

Cloning and characterization of the murine ameloblastin promoter.

The molecular mechanisms directing the highly restricted expression pattern of murine ameloblastin were characterized by cloning and functional analysis of the ameloblastin promoter. The transcription start site, mapped by primer extension, was located 19 base pairs (bp) 5' of the published cDNA. The promoter was analyzed in a mouse ameloblast-like cell line (LS8) and was compared with promoter activity in primary gingival fibroblasts and pulp fibroblasts. Sequential 5'-deletion mutants encompassing DNA sequences from -1616 to -781 bp exhibited high promoter activity in LS8 cells, whereas the promoter activity decreased to 50% of the full-length construct in the -781- and -477-bp regions. The -217-bp promoter region regained promoter activity that approached the activity of the full-length promoter construct, suggesting that both positive and negative cis-acting regions may be involved in ameloblastin transcriptional regulation. Activity of the ameloblastin promoter in gingival and pulp fibroblasts was minimal and ranged from 8 to 30% of the activity in ameloblast-like cells. Several DNA-protein complexes were formed between functionally important promoter fragments and nuclear extracts from LS8 cells. The inactivity of promoter constructs in pulp and gingival fibroblasts as well as the absence of similar DNA-protein complexes from these cells suggest that regulatory regions of the murine ameloblastin promoter may function in a cell-specific manner.

Animals↗
PubMed1999

Bone marrow stromal cells: characterization and clinical application.

The bone marrow stroma consists of a heterogeneous population of cells that provide the structural and physiological support for hematopoietic cells. Additionally, the bone marrow stroma contains cells with a stem-cell-like character that allows them to differentiate into bone, cartilage, adipocytes, and hematopoietic supporting tissues. Several experimental approaches have been used to characterize the development and functional nature of these cells in vivo and their differentiating potential in vitro. In vivo, presumptive osteogenic precursors have been identified by morphologic and immunohistochemical methods. In culture, the stromal cells can be separated from hematopoietic cells by their differential adhesion to tissue culture plastic and their prolonged proliferative potential. In cultures generated from single-cell suspensions of marrow, bone marrow stromal cells grow in colonies, each derived from a single precursor cell termed the colony-forming unit-fibroblast. Culture methods have been developed to expand marrow stromal cells derived from human, mouse, and other species. Under appropriate conditions, these cells are capable of forming new bone after in vivo transplantation. Various methods of cultivation and transplantation conditions have been studied and found to have substantial influence on the transplantation outcome. The finding that bone marrow stromal cells can be manipulated in vitro and subsequently form bone in vivo provides a powerful new model system for studying the basic biology of bone and for generating models for therapeutic strategies aimed at regenerating skeletal elements.

Adipocytes↗
PubMed1999

Isolation and characterization of MC3T3-E1 preosteoblast subclones with distinct in vitro and in vivo differentiation/mineralization potential.

A series of subclonal cell lines with high or low differentiation/mineralization potential after growth in the presence of ascorbic acid (AA) were derived from murine MC3T3-E1 cells. Subclones were characterized in terms of their ability to mineralize a collagenous extracellular matrix both in vitro and in vivo and express osteoblast-related genes. When compared with nonmineralizing cells, mineralizing subclones selectively expressed mRNAs for the osteoblast markers, bone sialoprotein (BSP), osteocalcin (OCN), and the parathyroid hormone (PTH)/parathyroid hormone-related protein (PTHrP) receptor. In contrast, alkaline phosphatase mRNA was present in certain nonmineralizing as well as mineralizing subclones, suggesting that its expression may be subject to different controls from other osteoblast markers. Only highly differentiating subclones exhibited strong AA-dependent induction of a transiently transfected OCN promoter-luciferase reporter gene, indicating that there was a good correlation between mRNA levels and transcriptional activity. Consistent with its postulated role in biomineralization, BSP as measured by Western blotting was only present in mineralizing subclones. After implantation into immunodeficient mice, highly differentiating subclones formed bone-like ossicles resembling woven bone, while poorly differentiating cells only produced fibrous tissue. Interestingly, subclones with both high and low differentiation potential produced similar amounts of collagen in culture and expressed comparable basal levels of mRNA encoding Osf2/Cbfa1, an osteoblast-related transcription factor. Although some strongly differentiating cells exhibited a modest AA-dependent up-regulation of Osf2/Cbfa1 mRNA, there was no clear relationship between levels of this message and induction of mRNAs for other differentiation markers. Thus, the mere presence of Osf2/Cbfa1 in a subclone was not sufficient for osteoblast differentiation. These subclones will be very useful for studying critical events in osteoblast differentiation and mineralization.

Animals↗
PubMed1998

Repair of craniotomy defects using bone marrow stromal cells.

BACKGROUND: Techniques used to repair craniofacial skeletal defects parallel the accepted surgical therapies for bone loss elsewhere in the skeleton and include the use of autogenous bone and alloplastic materials. Transplantation of a bone marrow stromal cell population that contains osteogenic progenitor cells may be an additional modality for the generation of new bone. METHODS: Full thickness osseous defects (5 mm) were prepared in the cranium of immunocompromised mice and were treated with gelatin sponges containing murine alloplastic bone marrow stromal cells derived from transgenic mice carrying a type I collagen-chloramphenicol acetyltransferase reporter gene to follow the fate of the transplanted cells. Control surgical sites were treated with spleen stromal cells or gelatin sponges alone, or were left untreated. The surgical defects were analyzed histologically for percent closure of the defect at 2, 3, 4, 6, and 12 weeks. RESULTS: Cultured bone marrow stromal cells transplanted within gelatin sponges resulted in osteogenesis that repaired greater than 99.0+/-2.20% of the original surgical defect within 2 weeks. In contrast, cranial defects treated with splenic fibroblasts, vehicle alone, or sham-operated controls resulted in minimal repair that was limited to the surgical margins. Bone marrow stromal cells carrying the collagen transgene were immunodetected only in the newly formed bone and thus confirmed the donor origin of the transplanted cells. CONCLUSIONS: These studies demonstrate that mitotically expanded bone marrow cells can serve as an abundant source of osteoprogenitor cells that are capable of repairing craniofacial skeletal defects in mice without the addition of growth or morphogenetic factors.

Animals↗