Paul H. Krebsbach

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.

Evidence for tissue-resident mesenchymal stem cells in human adult lung from studies of transplanted allografts

The origin and turnover of connective tissue cells in adult human organs, including the lung, are not well understood. Here, studies of cells derived from human lung allografts demonstrate the presence of a multipotent mesenchymal cell population, which is locally resident in the human adult lung and has extended life span in vivo. Examination of plastic-adherent cell populations in bronchoalveolar lavage samples obtained from 76 human lung transplant recipients revealed clonal proliferation of fibroblast-like cells in 62% (106 of 172) of samples. Immunophenotyping of these isolated cells demonstrated expression of vimentin and prolyl-4-hydroxylase, indicating a mesenchymal phenotype. Multiparametric flow cytometric analyses revealed expression of cell-surface proteins, CD73, CD90, and CD105, commonly found on mesenchymal stem cells (MSCs). Hematopoietic lineage markers CD14, CD34, and CD45 were absent. Multipotency of these cells was demonstrated by their capacity to differentiate into adipocytes, chondrocytes, and osteocytes. Cytogenetic analysis of cells from 7 sex-mismatched lung transplant recipients harvested up to 11 years after transplant revealed that 97.2% +/- 2.1% expressed the sex genotype of the donor. The presence of MSCs of donor sex identity in lung allografts even years after transplantation provides what we believe to be the first evidence for connective tissue cell progenitors that reside locally within a postnatal, nonhematopoietic organ.

Endothelial cell modulation of bone marrow stromal cell osteogenic potential

In the context of bone development and regeneration, the intimate association of the vascular endothelium with osteogenic cells suggests that endothelial cells (ECs) may directly regulate the differentiation of osteoprogenitor cells. To investigate this question, bone marrow stromal cells (BMSCs) were cultured: in the presence of EC‐conditioned medium, on EC extracellular matrix, and in EC cocultures with and without cell contact. RNA and protein were isolated from ECs and analyzed by reverse transcriptase‐polymerase chain reaction and Western blotting, respectively, for expression of bone morphogenetic protein 2 (BMP‐2). In animal studies, BMSCs and ECs were cotransplanted into severe combined immunodeficient mice on biodegradable polymer matrices, and histomorphometric analysis was performed to determine the extent of new bone and blood vessel formation. ECs significantly increased BMSC osteogenic differentiation in vitro only when cultured in direct contact. ECs expressed BMP‐2, and experiments employing interfering RNA inhibition confirmed its production as contributing to the increased BMSC osteogenic differentiation. In vivo, cotransplantation of ECs with BMSCs resulted in greater bone formation than did transplantation of BMSCs alone. These data suggest that ECs function not only to form the microvasculature that delivers nutrients to developing bone but also to modulate the differentiation of osteoprogenitor cells in vitro and in vivo.

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 (μCT) was used to assess mineralized bone matrix. Though the magnitude of the angiogenic response between groups was the same, μCT 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.

Gene Therapy Approaches for Bone Regeneration

Gene therapy represents a promising approach for delivering regenerative molecules to specific tissues including bone. Several laboratories have shown that virus-based BMP expression vectors can stimulate osteoblast differentiation and bone formation in vivo. Both in vivo and ex vivo transduction of cells can induce bone formation at ectopic and orthotopic sites. Adenovirus and direct DNA delivery of genes encoding regenerative molecules can heal critical-sized defects of cranial and long bones. Although osteogenic activity can be demonstrated for individual BMP vectors, substantial synergies may be achieved using combinatorial gene therapy to express complimentary osteogenic signals including specific combinations of BMPs or BMPs and transcription factors. Further control of the bone regeneration process may also be achieved through the use of inducible promoters that can be used to control the timing and magnitude of expression for a particular gene. Using these types of approaches, it should be possible to mimic natural processes of bone development and fracture repair and, in so doing, be able to precisely control both the amount and type of bone regenerated.

Erythropoietin Couples Hematopoiesis with Bone Formation

Background It is well established that bleeding activates the hematopoietic system to regenerate the loss of mature blood elements. We have shown that hematopoietic stem cells (HSCs) isolated from animals challenged with an acute bleed regulate osteoblast differentiation from marrow stromal cells. This suggests that HSCs participate in bone formation where the molecular basis for this activity is the production of BMP2 and BMP6 by HSCs. Yet, what stimulates HSCs to produce BMPs is unclear. Methodology/Principal Findings In this study, we demonstrate that erythropoietin (Epo) activates Jak-Stat signaling pathways in HSCs which leads to the production of BMPs. Critically, Epo also directly activates mesenchymal cells to form osteoblasts in vitro, which in vivo leads to bone formation. Importantly, Epo first activates osteoclastogenesis which is later followed by osteoblastogenesis that is induced by either Epo directly or the expression of BMPs by HSCs to form bone. Conclusions/Significance These data for the first time demonstrate that Epo regulates the formation of bone by both direct and indirect pathways, and further demonstrates the exquisite coupling between hematopoesis and osteopoiesis in the marrow.

Resident Tissue-Specific Mesenchymal Progenitor Cells Contribute to Fibrogenesis in Human Lung Allografts

Fibrotic obliteration of the small airways leading to progressive airflow obstruction, termed bronchiolitis obliterans syndrome (BOS), is the major cause of poor outcomes after lung transplantation. We recently demonstrated that a donor-derived population of multipotent mesenchymal stem cells (MSCs) can be isolated from the bronchoalveolar lavage (BAL) fluid of human lung transplant recipients. Herein, we study the organ specificity of these cells and investigate the role of local mesenchymal progenitors in fibrogenesis after lung transplantation. We demonstrate that human lung allograft-derived MSCs uniquely express embryonic lung mesenchyme-associated transcription factors with a 35,000-fold higher expression of forkhead/winged helix transcription factor forkhead box (FOXF1) noted in lung compared with bone marrow MSCs. Fibrotic differentiation of MSCs isolated from normal lung allografts was noted in the presence of profibrotic mediators associated with BOS, including transforming growth factor-β and IL-13. MSCs isolated from patients with BOS demonstrated increased expression of α-SMA and collagen I when compared with non-BOS controls, consistent with a stable in vivo fibrotic phenotype. FOXF1 mRNA expression in the BAL cell pellet correlated with the number of MSCs in the BAL fluid, and myofibroblasts present in the fibrotic lesions expressed FOXF1 by in situ hybridization. These data suggest a key role for local tissue-specific, organ-resident, mesenchymal precursors in the fibrogenic processes in human adult lungs.

Processing of Ameloblastin by MMP-20

Ameloblastin (AMBN) cleavage products are the most abundant non-amelogenin proteins in the enamel matrix of developing teeth. AMBN N-terminal cleavage products accumulate in the sheath space between enamel rods, while AMBN C-terminal products localize within rods. We tested the hypothesis that MMP-20 is the protease that cleaves AMBN. Glycosylated recombinant porcine AMBN (rpAMBN) was expressed in human kidney 293F cells, and recombinant porcine enamelysin (rpMMP-20) was expressed in bacteria. The purified proteins were incubated together at an enzyme:substrate ratio of 1:100. N-terminal sequencing of AMBN digestion products determined that rpMMP-20 cleaved rpAMBN after Pro2, Gln130, Gln139, Arg170, and Ala222. This shows that MMP-20 generates the 23-kDa AMBN starting at Tyr223, as well as the 17-kDa (Val1-Arg170) and 15-kDa (Val1-Gln130) AMBN cleavage products that concentrate in the sheath space during the secretory stage. We conclude that MMP-20 processes ameloblastin in vitro and in vivo.

Macro-Architectures in Spinal Cord Scaffold Implants Influence Regeneration

Biomaterial scaffold architecture has not been investigated as a tunable source of influence on spinal cord regeneration. This study compared regeneration in a transected spinal cord within various designed-macro-architecture scaffolds to determine if these architectures alone could enhance regeneration. Three-dimensional (3-D) designs were created and molds were built on a 3-D printer. Salt-leached porous poly(epsilon-caprolactone) was cast in five different macro-architectures: cylinder, tube, channel, open-path with core, and open-path without core. The two open-path designs were created in this experiment to compare different supportive aspects of architecture provided by scaffolds and their influence on regeneration. Rats received T8 transections and implanted scaffolds for 1 and 3 months. Overall morphology and orientation of sections were characterized by H&E, luxol fast blue, and cresyl violet staining. Borders between intact gray matter and non-regenerated defect were observed from GFAP immunolabeling. Nerve fibers and regenerating axons were identified with Tuj-1 immunolabeling. The open-path designs allowed extension of myelinated fibers along the length of the defect both exterior to and inside the scaffolds and maintained their original defect length up to 3 months. In contrast, the cylinder, tube, and channel implants had a doubling of defect length from secondary damage and large scar and cyst formation with no neural tissue bridging. The open-path scaffold architectures enhanced spinal cord regeneration compared to the three other designs without the use of biological factors.

Epithelial Interactions and Local Engraftment of Lung-Resident Mesenchymal Stem Cells

Multipotent mesenchymal progenitor cells, termed mesenchymal stem cells (MSCs), have been demonstrated to reside in human adult lungs. However, there is little information regarding the associations of these local mesenchymal progenitors with other resident somatic cells and their potential for therapeutic use. Here we provide in vivo and in vitro evidence for the ability of human adult lung-resident MSCs (LR-MSCs) to interact with the local epithelial cells. The in vivo retention and localization of human LR-MSCs in an alveolar microenvironment was investigated by placing PKH-26 or DsRed lentivirus-labeled human LR-MSCs in the lungs of immunodeficient (SCID) mice. At 3 weeks after intratracheal administration, 19.3 ± 3.21% of LR-MSCs were recovered, compared with 3.47 ± 0.51% of control fibroblasts, as determined by flow cytometry. LR-MSCs were found to persist in murine lungs for up to 6 months and demonstrated preferential localization to the corners of the alveoli in close proximity to type II alveolar epithelial cells, the progenitor cells of the alveolar epithelium. In vitro, LR-MSCs established gap junction communications with lung alveolar and bronchial epithelial cells and demonstrated an ability to secrete keratinocyte growth factor, an important modulator of epithelial cell proliferation and differentiation. Gap junction communications were also demonstrable between LR-MSCs and resident murine cells in vivo. This study demonstrates, for the first time, an ability of tissue-specific MSCs to engraft in their organ of origin and establishes a pathway of bidirectional interaction between these mesenchymal progenitors and adult somatic epithelial cells in the lung.