Transplantation of Human Mesenchymal Stems Cells Into Intervertebral Discs in a Xenogeneic Porcine Model (original) (raw)
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Joint Bone Spine, 2009
Objectives: Candidate cell types for disc cell transplantation therapy include anulus fibrosus (AF) cells, chondrocytes, and bone marrow derived cells (BMDCs). We compared the disc matrix production in these three types of cells, before and after stimulation with rhBMP-2. There is no study extant that compares these three cell types to determine the best candidate for the disc cell therapy. Methods: AF cells, chondrocytes, and BMDCs (iliac crest and femur) were isolated and grown in monolayer. They were treated for 3 days with rhBMP-2. After 3 days, proteoglycan (sGAG) content in the media was quantified. The results were normalized by cell numbers. The mRNA expression of aggrecan, type I collagen, and type II collagen was measured using real-time PCR.
JOR Spine
Background: Forty percent of low back pain cases are due to intervertebral disc degeneration (IVDD), with mesenchymal stem cells (MSCs) a reported treatment. We utilized an ovine IVDD model and intradiscal heterologous MSCs to determine therapeutic efficacy at different stages of IVDD. Methodology: Three nonoperated control (NOC) sheep were used for MSC isolation. In 36 sheep, 6 × 20 mm annular lesions were made at three spinal levels using customized blades/ scalpel handles, and IVDD was allowed to develop for 4 weeks in the Early (EA) and late Acute (LA) groups, or 12 weeks in the chronic (EST) group. Lesion IVDs received injections of 10 × 10 6 MSCs or PBS, and after 8 (EA), 22 (LA) or 14 (EST) weeks recuperation the sheep were sacrificed. Longitudinal lateral radiographs were used to determine disc heights. IVD glycosaminoglycan (GAG) and hydroxyproline contents were quantified using established methods. An Instron materials testing machine and customized jigs analyzed IVD (range of motion, neutral zone [NZ] and stiffness) in flexion/extension, lateral bending and axial rotation. qRTPCR gene profiles of key anabolic and catabolic matrix molecules were undertaken. Toluidine blue and hematoxylin and eosin stained IVD sections were histopathologically scoring by two blinded observers. Results: IVDD significantly reduced disc heights. MSC treatment restored 95% to 100% of disc height, maximally improved NZ and stiffness in flexion/extension and lateral bending in the EST group, restoring GAG levels. With IVDD qRTPCR demonstrated elevated catabolic gene expression (MMP2/3/9/13, ADAMTS4/5) in the PBS IVDs and expession normalization in MSC-treated IVDs. Histopathology degeneracy scores were close to levels of NOC IVDs in MSC IVDs but IVDD developed in PBS injected IVDs. Discussion: Administered MSCs produced recovery in degenerate IVDs, restored disc height, composition, biomechanical properties, down regulated MMPs and fibrosis, strongly supporting the efficacy of MSCs for disc repair. K E Y W O R D S intervertebral disc, intervertebral disc degeneration, intervertebral disc repair, mesenchymal stem cells 1 | INTRODUCTION The IVD is a fibrocartilaginous visco-elastic weight bearing cushion that provides mechanical stability and spinal flexibility during axial loading, flexion and rotation. 1-3 The IVD is composed of superior and inferior cartilagenous end plates (CEPs) which interface with the vertebrae, the outer region of the disc, the annulus fibrosus (AF) is a collagen rich tissue which provides mechanical strength during stretching
Tissue Engineering Part A, 2009
This study aimed to evaluate whether rat mesenchymal stem cells (rMSCs) could be differentiated in vitro into disc-like cells by coculturing with intervertebral disc tissue. rMSCs were cultured with rodent intervertebral disc for up to 30 days in transwell plates. The differentiation of rMSCs was evaluated by immunostaining, Western blot, real-time RT-PCR, Northern blot, and electron microscopy. The potentials of multilineage differentiation and proteoglycan and collagen synthesis were also investigated. rMSCs underwent morphological changes to form three-dimensional micromasses and expressed collagen-2, aggrecan, and sox-9 at RNA and protein levels after 14 days of coculture. These changes were not detected in the samples of rMSCs cultured alone. Cocultured rMSCs also showed other characteristic features of disc-like cells, including the extracellular matrix formation, and proteoglycan and collagen synthesis. In addition, cellular contact between cocultured rMSCs and disc tissue was observed by electron microscopy. Committed rMSCs still retained their differentiation ability into mesoderm lineages of adipocytes or osteocytes when the local environment was altered. This study supports that MSCs are a promising source for cell therapy and tissue engineering in disc regeneration, and highlights that rMSCs can be induced into nucleus pulposus-like cells in vitro under the direct influence of intact disc tissue.
Human Umbilical Cord Blood–Derived Mesenchymal Stem Cells in the Cultured Rabbit Intervertebral Disc
American Journal of Physical Medicine & Rehabilitation, 2013
Objective-Back pain associated with symptomatic disc degeneration is a common clinical condition. Intervertebral disc (IVD) cell apoptosis and senescence increase with aging and degeneration. Repopulating the IVD with cells that could produce and maintain extracellular matrix would be an alternative therapy to surgery. The objective of this study was to determine the potential of human umbilical cord blood-derived mesenchymal stem cells (hUCB-MSCs) as a novel cell source for disc repair. In this study, we intended to confirm the potential for hUCB-MSCs to differentiate and display a chondrocyte-like phenotype after culturing in micromass and after injection into the rabbit IVD explant culture. We also wanted to confirm hUCB-MSC survival after transplantation into the IVD explant culture. Design-This study consisted of micromass cultures and in vitro rabbit IVD explant cultures to assess hUCB-MSC survival and differentiation to display chondrocyte-like phenotype. First, hUCB-MSCs were cultured in micromass and stained with Alcian blue dye. Second, to confirm cell survival, hUCB-MSCs were labeled with an infrared dye and a fluorescent dye before injection into whole rabbit IVD explants (host). IVD explants were then cultured for 4 wks. Cell survival was confirmed by two independent techniques: an imaging system detecting the infrared dye at the organ level and fluorescence microscopy detecting fluorescent dye at the cellular level. Cell viability was assessed by staining the explant with CellTracker green, a membrane-permeant
Influence of Porcine Intervertebral Disc Matrix on Stem Cell Differentiation
2011
For back disorders, cell therapy is one approach for a real regeneration of a degenerated nucleus pulposus. Human mesenchymal stem cells (hMSC) could be differentiated into nucleus pulposus (NP)-like cells and used for cell therapy. Therefore it is necessary to find a suitable biocompatible matrix, which supports differentiation. It could be shown that a differentiation of hMSC in a microbial transglutaminase cross-linked gelatin matrix is possible, but resulted in a more chondrocyte-like cell type. The addition of porcine NP extract to the gelatin matrix caused a differentiation closer to the desired NP cell phenotype. This concludes that a hydrogel containing NP extract without any other supplements could be suitable for differentiation of hMSCs into NP cells. The NP extract itself can be cross-linked by transglutaminase to build a hydrogel free of NP atypical substrates. As shown by side-specific biotinylation, the NP extract contains molecules with free glutamine and lysine residues available for the transglutaminase.
International Journal of Molecular Sciences
The purpose of the present pilot study was to evaluate the effect of a hydrogel composed of hyaluronic acid (HA) and platelet-rich plasma (PRP) as a carrier for human mesenchymal stem cells (hMSCs) for intervertebral disc (IVD) regeneration using a disc organ culture model. HA was mixed with batroxobin (BTX) and PRP to form a hydrogel encapsulating 1 × 106 or 2 × 106 hMSCs. Bovine IVDs were nucleotomized and filled with hMSCs suspended in ~200 μL of the PRP/HA/BTX hydrogel. IVDs collected at day 0 and nucleotomized IVDs with no hMSCs and/or hydrogel alone were used as controls. hMSCs encapsulated in the hydrogel were also cultured in well plates to evaluate the effect of the IVD environment on hMSCs. After 1 week, tissue structure, scaffold integration, hMSC viability and gene expression of matrix and nucleus pulposus (NP) cell markers were assessed. Histological analysis showed a better preservation of the viability of the IVD tissue adjacent to the gel in the presence of hMSCs (~7...
European Spine Journal, 2012
Purpose Disc degeneration, and associated low back pain, are a primary cause of disability. Disc degeneration is characterized by dysfunctional cells and loss of proteoglycans: since intervertebral tissue has a limited capacity to regenerate, this process is at present considered irreversible. Recently, cell therapy has been suggested to provide more successful treatment of IVD degeneration. To understand the potential of cells to restore IVD structure/function, tissue samples from degenerated IVD versus healthy discs have been compared. Methods Discal tissue from 27 patients (40.17 ± 11 years) undergoing surgery for degenerative disc disease (DDD), DDD ? herniation and congenital scoliosis, as controls, was investigated. Cells and matrix in the nucleus pulposus (NP) and annulus fibrosus (AF) were characterized by histology. AF-and NP-derived cells were isolated, expanded and characterized for senescence and gene expression. Three-dimensional NP pellets were cultured and stained for glycosaminoglycan formation. Results Phenotypical markers of degeneration, such as cell clusters, chondrons, and collagen disorganization were seen in the degenerate samples. In severe degeneration, granulation tissue and peripheral vascularization were observed. No correlation was found between the Pfirrmann clinical score and the extent of degeneration. Conclusion The tissue disorganization in degenerate discs and the paucity of cells out of cluster/chondron association, make the IVD-derived cells an unreliable option for disc regeneration.