Edward J. Caterson

Human marrow-derived mesenchymal progenitor cells: isolation, culture expansion, and analysis of differentiation.

A number of adult mesenchymal tissues contain subpopulations of undifferentiated cells, which retain the capacity to differentiate along multiple lineages. These mesenchymal progenitor cells may be cultured in an undifferentiated state and, when given the appropriate signals, differentiate into an expanding list of several mesenchymal and a few ectodermal derived tissues. The maintenance and propagation of the multipotential nature of these progenitor cell populations are crucially dependent on the isolation protocol, the culture expansion conditions, particularly the properties of the fetal bovine serum supplement in the culture medium. This article describes a method for selection of the appropriate serum lot, and introduces a simplified isolation technique to optimize the yield of progenitor cells that maintain the capability of undergoing multilineage differentiation in response to appropriate cues. Cell populations isolated and culture expanded in this manner, by virtue of their multiple differentiation potential, should serve as ideal candidate cells for tissue engineering applications for the repair and regeneration of tissue damaged by disease and or trauma.

Three‐dimensional cartilage formation by bone marrow‐derived cells seeded in polylactide/alginate amalgam

Bone marrow-derived cells are considered as candidate cells for cartilage tissue engineering by virtue of their ability to undergo chondrogenesis in vitro when cultured in high density or when embedded within a three-dimensional matrix in the presence of growth factors. This study evaluated the potential of human bone marrow-derived cells for cartilage tissue engineering by examining their chondrogenic properties within a three-dimensional amalgam scaffold consisting of the biodegradable polymer, poly-L-lactic acid (PLA) alone, and with the polysaccharide gel, alginate. Cells were suspended either in alginate or medium and loaded into porous PLA blocks. Alginate was used to improve cell loading and retention within the construct, whereas the PLA polymeric scaffold provided appropriate mechanical support and stability to the composite culture. Cells seeded in the PLA/alginate amalgams and the plain PLA constructs were treated with different concentrations of recombinant human transforming growth factor-beta1 (TGF-beta 1) either continuously (10 ng/mL) or only for the initial 3 days of culture (50 ng/mL). Chondrogenesis was assessed at weekly intervals with cultures maintained for up to 3 weeks. Histological and immunohistochemical analysis of the TGF-beta 1-treated PLA/alginate amalgam and PLA constructs showed development of a cartilaginous phenotype from day 7 to day 21 as demonstrated by colocalization of Alcian blue staining with collagen type II and cartilage proteoglycan link protein. Expression of cartilage specific genes, including collagen types II and IX, and aggrecan, was detected in TGF-beta 1-treated cultures by reverse transcription-polymerase chain reaction analysis. The initiation and progression of chondrogenic differentiation within the polymeric macrostructure occurred with both continuous and the initial 3-day TGF-beta 1 treatment regimens, suggesting that key regulatory events of chondrogenesis take place during the early period of cell growth and proliferation. Scanning electron microscopy revealed abundant cells with a rounded morphology in the PLA/alginate amalgam. These findings suggest that the three-dimensional PLA/alginate amalgam is a potential candidate bioactive scaffold for cartilage tissue engineering applications.

Three Patients with Full Facial Transplantation

Unlike conventional reconstruction, facial transplantation seeks to correct severe deformities in a single operation. We report on three patients who received full-face transplants at our institution in 2011 in operations that aimed for functional restoration by coaptation of all main available motor and sensory nerves. We enumerate the technical challenges and postoperative complications and their management, including single episodes of acute rejection in two patients. At 6 months of follow-up, all facial allografts were surviving, facial appearance and function were improved, and glucocorticoids were successfully withdrawn in all patients.

Medical 3D Printing for the Radiologist

While use of advanced visualization in radiology is instrumental in diagnosis and communication with referring clinicians, there is an unmet need to render Digital Imaging and Communications in Medicine (DICOM) images as three-dimensional (3D) printed models capable of providing both tactile feedback and tangible depth information about anatomic and pathologic states. Three-dimensional printed models, already entrenched in the nonmedical sciences, are rapidly being embraced in medicine as well as in the lay community. Incorporating 3D printing from images generated and interpreted by radiologists presents particular challenges, including training, materials and equipment, and guidelines. The overall costs of a 3D printing laboratory must be balanced by the clinical benefits. It is expected that the number of 3D-printed models generated from DICOM images for planning interventions and fabricating implants will grow exponentially. Radiologists should at a minimum be familiar with 3D printing as it relates to their field, including types of 3D printing technologies and materials used to create 3D-printed anatomic models, published applications of models to date, and clinical benefits in radiology. Online supplemental material is available for this article.

Polymer/Alginate Amalgam for Cartilage- Tissue Engineering

Abstract: Marrow stroma‐derived cells (MSC) are highly proliferative, multipotential cells that have been considered as ideal candidate cells for autologous tissue engineering applications. In this study, we have characterized the chondrogenic potential of human MSCs in both a PLA/alginate amalgam and pure PLA macrostructure as model three‐dimensional constructs to support both chondrogenic differentiation and proliferation following TGF‐β treatment. MSCs were seeded in experimental groups that consisted of PLA‐loaded constructs and PLA/alginate amalgams with and without recombinant human TGF‐β1. Chondrogenesis of the PLA and the PLA/alginate amalgam cultures was assessed at weekly intervals by histology, immunohistochemistry, scanning electron microscopy, sulfate incorporation, and RT‐PCR. Chondrogenic differentiation occurs within a polymeric macrostructure with TGF‐β1 treatment as indicated by histological, immunohistochemical, sulfate incorporation, and gene expression profiles. This macrostructure can be further encased in an alginate gel/solution to optimize cell shape and to confine growth factors and cells within the polymer construct, while the polymeric scaffold provides appropriate mechanical/tissue support. The stable three‐dimensional PLA/alginate amalgam represents a novel candidate system of mesenchymal chondrogenesis, which is amendable to investigation of mechanical and biological factors that normally modulate cartilage development and formation as well as a potential tissue engineering construct for cartilage repair.

Tissue Engineering of Skin

Tissue engineering (TE) and regenerative medicine area blend of developmental biology, life sciences, and engi-neering efforts that attempts to address clinical problems.Tissue engineering was defined in 1988 as the applicationof principles and methods of engineering and lifesciences toward fundamental understanding of structurefunction relationships in normal and pathologicalmammalian tissues and the development of biologicsubstitutes to restore, maintain, or improve tissue func-tion

TGF‐β1 calcium signaling in osteoblasts

Transforming growth factor‐β1 (TGF‐β1) action is known to be initiated by its binding to multiple cell surface receptors containing serine/threonine kinase domains that act to stimulate a cascade of signaling events in a variety of cell types. We have previously shown that TGF‐β1 and BMP‐2 treatment of primary human osteoblasts (HOBs) enhances cell‐substrate adhesion. In this report, we demonstrate that TGF‐β1 elicits a rapid, transient, and oscillatory rise in the intracellular Ca2+ concentration, [Ca2+]i, that is necessary for enhancement of cell adhesion in HOBs but does not alter the phosphorylation state of Smad proteins. This rise in [Ca2+]i in HOB is not observed in the absence of extracellular calcium or when the cells are treated with the L‐type Ca2+ channel blocker, nifedipine, but is stimulated upon treatment with the L‐type Ca2+ channel agonist, Bay K 8644, or under high K+ conditions. The rise in [Ca2+]i is severely attenuated after treatment of the cells with thapsigargin, a selective endoplasmic reticulum Ca2+ pump inhibitor. TGF‐β1 enhancement of HOB adhesion to tissue culture polystyrene is also inhibited in cells treated with nifedipine. These data suggest that intracellular Ca2+ signaling is an important second messenger of the TGF‐β1 signal transduction pathway in osteoblast function. J. Cell. Biochem. 101: 348–359, 2007.

Multilineage Differentiation of Adult Human Bone Marrow Progenitor Cells Transduced with Human Papilloma Virus Type 16 E6/E7 Genes

We have established a new adult human bone marrow-derived cell line hMPC 32F, stably transduced with human papilloma virus type 16 E6/E7 genes, that displays mesenchymal multilineage differentiation ability in vitro. The hMPC 32F cells exhibited a population doubling time of 22 h and have been maintained in culture for about 20 passages. When cultured in conditions promoting osteogenic, adipogenic, or chondrogenic differentiation, hMPC 32F cells expressed mature differentiated phenotypes. These include (1) osteoblastic phenotype characterized by upregulated alkaline phosphatase (ALP) expression and extracellular matrix mineralization, (2) adipocytic phenotype with the presence of intracellular lipid droplets, and (3) chondrocytic phenotype of round cells surrounded by a sulfated proteoglycan-rich matrix. In addition, the hMPC 32F cells expressed differentiation lineage-specific genes, as detected by RT-PCR. Furthermore, osteogenic and adipogenic cultures responded to regulatory factors such as transforming growth factor-b1 (TGF-b1) and 1a, 25-dihydroxyvitamin D3 (1,25(OH)2D3). Thus, continuous treatment of osteogenic cultures for 2 weeks with TGF-b1 decreased ALP activity and mRNA expression and inhibited osteocalcin mRNA expression and matrix mineralization, whereas l,25(OH)2D3 had an additive, stimulatory effect. In adipogenic cultures, treatment with TGF-b1 for 2 weeks markedly inhibited adipogenesis whereas 1,25(OH)2D3 had no obvious effect. Finally, clonal analysis of hMPC 32F cells revealed a high percentage of multipotent clones, although clones of more restricted differentiation potential were also present. These characteristics of the hMPC 32F cell line suggest their pluripotent, progenitor, and nontransformed nature and indicate their potential application for studying the mechanisms governing developmental potential of adult human bone marrow mesenchymal progenitor cells.

TGF-β1 calcium signaling increases α5 integrin expression in osteoblasts

TGF‐β1 is a potent osteoactive factor and exhibits a wide variety of effects on osteoblasts, most of which are mediated through receptor associated Smad proteins. We have recently reported a novel TGF‐β1 intracellular Ca2+ signaling pathway in osteoblasts, and found that this signaling is required for the TGF‐β1 mediated enhancement of osteoblast adhesion to substrate. Given that interaction between the extracellular matrix protein fibronectin and α5β1 integrin on the cell surface is principally responsible for osteoblast substrate adhesion, we examined here whether the TGF‐β1 stimulated Ca2+ signal is involved in this pathway. Our results show that, in primary human osteoblasts, the TGF‐β1 induced intracellular Ca2+ signal is responsible, in part, for the stimulation of expression of α5 integrin, but not of β1 integrin or fibronectin. Increased levels of α5 integrin protein and mRNA were seen as early as 12 h after TGF‐β1 treatment, but were inhibited by co‐treatment of cells with nifedipine, a selective L‐type Ca2+ channel blocker. TGF‐β1 treatment increased both fibronectin and β1 integrin protein production within 48 h, in a manner unaffected by co‐treatment with nifedipine.

Epidermal regeneration by micrograft transplantation with immediate 100-fold expansion.

Background: Major loss of skin following burns or trauma requires skin grafting for repair. In addition, chronic wounds frequently require skin grafts. Current treatments are either cumbersome, limited in possible expansion ratio, costly, or require extensive time for treatment. This study investigates a new way of regenerating skin after major burns and other trauma, providing 100-fold expansion of a split-thickness skin graft. Methods: Submillimeter micrografts were created by controlled mincing of a split-thickness skin graft and transplanted to porcine full-thickness wounds. By creating an incubator-like microenvironment using wound chambers, the micrografts provide reepithelialization whether placed dermal side up or dermal side down. Results: Transplantation of micrografts in a 1:100 expansion ratio results in complete epithelialization of both healthy and diabetic wounds within 14 days. In comparison, nontransplanted wounds showed 62 percent reepithelialization in healthy pigs and 49 percent in diabetic pigs at the corresponding time point. Conclusions: Minced skin micrografts are very effective in wound repair and can provide 100-fold expansion of a skin graft. Early clinical results confirm the utility of this technique.