Keith G. Danielson

Multilineage mesenchymal differentiation potential of human trabecular bone-derived cells.

Explant cultures of adult human trabecular bone fragments give rise to osteoblastic cells, that are known to express osteoblast‐related genes and mineralize extracellular matrix. These osteoblastic cells have also been shown to undergo adipogenesis in vitro and chondrogenesis in vivo. Here we report the in vitro developmental potential of adult human osteoblastic cells (hOB) derived from explant cultures of collagenase‐pretreated trabecular bone fragments. In addition to osteogenic and adipogenic differentiation, these cells are capable of chondrogenic differentiation in vitro in a manner similar to adult human bone marrow‐derived mesenchymal progenitor cells. High‐density pellet cultures of hOB maintained in chemically defined serum‐free medium, supplemented with transforming growth factor‐β1, were composed of morphologically distinct, chondrocyte‐like cells expressing mRNA transcripts of collagen types II, IX and X, and aggrecan. The cells within the high‐density pellet cultures were surrounded by a sulfated prote‐oglycan‐rich extracellular matrix that immunostained for collagen type II and proteoglycan link protein. Osteogenic differentiation of hOB was verified by an increased number of alkaline phosphatase‐positive cells, that expressed osteoblast‐related transcripts such as alkaline phosphatase, collagen type I, osteopontin and osteocalcin, and formed mineralized matrix in monolayer cultures treated with ascorbate, β‐glycerophosphate, and bone morphogenetic protein‐2. Adipogenic differentiation of hOB was determined by the appearance of intracellular lipid droplets, and expression of adipocyte‐specific genes, such as lipoprotein lipase and peroxisome proliferator‐activated receptor γ2, in monolayer cultures treated with dexamethasone, indomethacin, insulin and 3‐isobutyl‐l‐methylxanthine. Taken together, these results show that cells derived from collagenase‐treated adult human trabecular bone fragments have the potential to differentiate into multiple mesenchymal lineages in vitro, indicating their developmental plasticity and suggesting their mesenchymal progenitor nature.

Transforming Growth Factor-β-mediated Chondrogenesis of Human Mesenchymal Progenitor Cells Involves N-cadherin and Mitogen-activated Protein Kinase and Wnt Signaling Cross-talk

The multilineage differentiation potential of adult tissue-derived mesenchymal progenitor cells (MPCs), such as those from bone marrow and trabecular bone, makes them a useful model to investigate mechanisms regulating tissue development and regeneration, such as cartilage. Treatment with transforming growth factor-β (TGF-β) superfamily members is a key requirement for the in vitro chondrogenic differentiation of MPCs. Intracellular signaling cascades, particularly those involving the mitogen-activated protein (MAP) kinases, p38, ERK-1, and JNK, have been shown to be activated by TGF-βs in promoting cartilage-specific gene expression. MPC chondrogenesis in vitro also requires high cell seeding density, reminiscent of the cellular condensation requirements for embryonic mesenchymal chondrogenesis, suggesting common chondro-regulatory mechanisms. Prompted by recent findings of the crucial role of the cell adhesion protein, N-cadherin, and Wnt signaling in condensation and chondrogenesis, we have examined here their involvement, as well as MAP kinase signaling, in TGF-β1-induced chondrogenesis of trabecular bone-derived MPCs. Our results showed that TGF-β1 treatment initiates and maintains chondrogenesis of MPCs through the differential chondro-stimulatory activities of p38, ERK-1, and to a lesser extent, JNK. This regulation of MPC chondrogenic differentiation by the MAP kinases involves the modulation of N-cadherin expression levels, thereby likely controlling condensation-like cell-cell interaction and progression to chondrogenic differentiation, by the sequential up-regulation and progressive down-regulation of N-cadherin. TGF-β1-mediated MAP kinase activation also controls WNT-7A gene expression and Wnt-mediated signaling through the intracellular β-catenin-TCF pathway, which likely regulates N-cadherin expression and subsequent N-cadherin-mediated cell-adhesion complexes during the early steps of MPC chondrogenesis.

Characterization of Multipotential Mesenchymal Progenitor Cells Derived from Human Trabecular Bone

The in vitro culture of human trabecular bone‐derived cells has served as a useful system for the investigation of the biology of osteoblasts. The recent discovery in our laboratory of the multilineage mesenchymal differentiation potential of cells derived from collagenase‐treated human trabecular bone fragments has prompted further interest in view of the potential application of mesenchymal progenitor cells (MPCs) in the repair and regeneration of tissue damaged by disease or trauma. Similar to human MPCs derived from bone marrow, a clearer understanding of the variability associated with obtaining these bone‐derived cells is required in order to optimize the design and execution of applicable studies. In this study, we have identified the presence of a CD73+, STRO‐1+, CD105+, CD34−, CD45−, CD144− cell population resident within collagenase‐treated, culture‐processed bone fragments, which upon migration established a homogeneous population of MPCs. Additionally, we have introduced a system of culturing these MPCs that best supports and maintains their optimal differentiation potential during long‐term culture expansion. When cultured as described, the trabecular bone‐derived cells display stem cell‐like capabilities, characterized by a stable undifferentiated phenotype as well as the ability to proliferate extensively while retaining the potential to differentiate along the osteoblastic, adipocytic, and chondrocytic lineages, even when maintained in long‐term in vitro culture.

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.

Evidence for skeletal progenitor cells in the degenerate human intervertebral disc.

Study Design. To identify and characterize endogenous progenitor cell population from intervertebral disc. Objective. To determine if progenitor cells exist in degenerate human discs. Summary of Background Data. Back pain, a significant source of morbidity in our society, is directly linked to the pathology of the intervertebral disc. Because disc disease is accompanied by a loss of cellularity, there is considerable interest in regeneration of cells of both the anulus fibrosus (AF) and nucleus pulposus (NP). Methods. To determine if skeletal progenitor cells are present in the disc, samples were obtained from the degenerate AF and NP of 5 patients (Thompson grade 2 and 3, mean age 34 ± 7.6 years) undergoing anterior cervical discectomy and fusion procedures as well as adult rat lumbar spine. Results. Cells isolated from degenerate human tissues expressed CD105, CD166, CD63, CD49a, CD90, CD73, p75 low affinity nerve growth factor receptor, and CD133/1, proteins that are characteristic of marrow mesenchymal stem cells. In osteogenic media, there was an induction of alkaline phosphatase activity and expression of alkaline phosphatase, osteocalcin, and Runx-2 mRNA. When maintained in adipogenic media, a small percentage of cells displayed evidence of adipogenic differentiation: accumulation of cytosolic lipid droplets and increased expression of peroxisome proliferator-activated receptor-&ggr;2 and lipoporotein lipase mRNA. AF- and NP-derived cells also evidenced chondrogenic differentiation. CD133 (+) cells in the AF were able to commit to either the chondrogenic or adipogenic lineages. The results of the human disc studies were confirmed using cell derived from the NP and AF tissue of the mature rat disc. Conclusion. The analytical data indicated that the pathologically degenerate human disc contained populations of skeletal progenitor cells. These findings suggest that these endogenous progenitors may be used to orchestrate the repair of the intervertebral disc.

Nucleus pulposus cells express HIF‐1α under normoxic culture conditions: A metabolic adaptation to the intervertebral disc microenvironment

Nucleus pulposus (NP) cells of the intervertebral disc reside in an environment that has a limited vascular supply and generate energy through anaerobic glycolysis. The goal of the present study was to examine the expression and regulation of HIF‐1α, a transcription factor that regulates oxidative metabolism in nucleus pulposus cells. Nucleus pulposus cells were isolated from rat, human, and sheep disc and maintained at either 21% or 2% oxygen for various time periods. Cells were also treated with desferrioxamine (Dfx), a compound that mimics the effects of hypoxia (Hx). Expression and function of HIF‐1α were assessed by immunofluorescence microscopy, Western blot analysis, gel shift assays, and luciferase reporter assays. In normoxia (Nx), rat, sheep, and human nucleus pulposus cells consistently expressed the HIF‐1α subunit. Unlike other skeletal cells, when maintained under low oxygen tension, the nucleus pulposus cells exhibited a minimal induction in HIF‐1α protein levels. Electromobility shift assays confirmed the functional binding of normoxic HIF‐1α protein to its putative DNA binding motif. A dual luciferase reporter assay showed increased HIF‐1α transcriptional activity under hypoxia compared to normoxic level, although this induction was small when compared to HeLa and other cell types. These results indicate that normoxic stabilization of HIF‐1α is a metabolic adaptation of nucleus pulposus cells to a unique oxygen‐limited microenvironment. The study confirmed that HIF‐1α can be used as a phenotypic marker of nucleus pulposus cells. J. Cell. Biochem. 98: 152–159, 2006.

Titanium particles suppress expression of osteoblastic phenotype in human mesenchymal stem cells

Long‐term stability of arthroplasty prosthesis depends on the integration between osseous tissue and the implant biomaterial. Integrity of the osseous tissue requires the contribution of mesenchymal stem cells and their continuous differentiation into an osteoblastic phenotype. This study aims to investigate the hypothesis that exposure to wear debris particles derived from orthopaedic biomaterials affects the osteoblastic differentiation of human mesenchymal stem cells (hMSC). Upon in vitro culture in the presence of osteogenic supplements (OS), we observe that cultures of hMSCs isolated from femoral head bone marrow are capable of osteogenic differentiation, expressing alkaline phosphatase, osteocalcin, and bone sialoprotein (BSP), in addition to producing collagen type I and BSP accompanied by extracellular matrix mineralization. Exposure of OS‐treated hMSCs to submicron commercially pure titanium (cpTi) particles suppresses BSP gene expression, reduces collagen type I and BSP production, decreases cellular proliferation and viability, and inhibits matrix mineralization. In comparison, exposure to zirconium oxide (ZrO2) particles of similar size did not alter osteoblastic gene expression and resulted in only a moderate decrease in cellular proliferation and mineralization. Confocal imaging of cpTi‐treated hMSC cultures revealed patchy groups of cells displaying disorganized cyto‐skeletal architecture and low levels of extracellular BSP. These in vitro findings suggest that chronic exposure of marrow cells to titanium wear debris in vivo may contribute to decreased bone formation at the bone/implant interface by reducing the population of viable hMSCs and compromising their differentiation into functional osteoblasts. Understanding the nature of hMSC bioreactivity to orthopaedic wear debris should provide additional insights into mechanisms underlying aseptic loosening.

Structural and functional characterization of the human perlecan gene promoter. Transcriptional activation by transforming growth factor-beta via a nuclear factor 1-binding element.

Perlecan, a modular heparan sulfate proteoglycan of basement membranes and cell surfaces, plays a crucial role in regulating the assembly of extracellular matrices and the binding of nutrients and growth factors to target cells. To achieve a molecular understanding of perlecan gene regulation, we isolated the 5′-flanking region and investigated its functional promoter activity and its response to cytokines. Transient cell transfection assays, using plasmid constructs harboring the perlecan promoter linked to the chloramphenicol acetyltransferase reporter gene, demonstrated that the largest ∼2.5-kilobase construct contained maximal promoter activity. This promoter region was functionally active in a variety of cells of diverse histogenetic origin, thus corroborating the widespread expression of this gene product. Stepwise 5′ deletion analyses demonstrated that the −461-base pair (bp) proximal promoter retained ∼90% of the total activity, and internal deletions confirmed that the most proximal sequence was essential for proper promoter activity. Nanomolar amounts of transforming growth factor-β induced 2-3-fold perlecan mRNA and protein core levels in normal human skin fibroblasts, and this induction was transcriptionally regulated; in contrast, tumor necrosis factor-α had no effect and was incapable of counteracting the effects of TGF-β. Using additional 5′ deletions and DNase footprinting analyses, we mapped the TGF-β responsive region to a sequence of 177 bp contained between −461 and −285. This region harbored a 14-bp element similar to a TGF-β-responsive element present in the promoters of collagen α1(I), α2(I), elastin, and growth hormone. Electrophoretic mobility shift assays and mutational analyses demonstrated that the perlecan TGF-β-responsive element bound specifically to TGF-β-inducible nuclear proteins with high affinity for NF-1 member(s) of transcription factors.

A Simple, High-Yield Method for Obtaining Multipotential Mesenchymal Progenitor Cells from Trabecular Bone

In vitro cultures of primary, human trabecular bone-derived cells represent a useful system for investigation of the biology of osteoblasts. Our recent discovery of the multilineage mesenchymal differentiation potential of trabecular bone-derived cells suggests the potential application of these cells as mesenchymal progenitors for tissue repair and regeneration. Such applications are crucially dependent on efficient cellisolation protocols to yield cells that optimally proliferate and differentiate. In this study, we describe a simple, high-yield procedure, requiring minimal culture expansion, for the isolation of mesenchymal progenitor cells from human trabecular bone. Moreover, these cells retain their ability to differentiate along multiple mesenchymal lineages through successive subculturing. Cell populations isolated and cultured as described here allow the efficient acquisition of a clinically significant number of cells, which may be used as the cell source for tissue-engineering applications.