Understanding and leveraging cell metabolism to enhance mesenchymal stem cell transplantation survival in tissue engineering and regenerative medicine applications.

In tissue engineering and regenerative medicine, stem cell-specifically, mesenchymal stromal/stem cells (MSCs)-therapies have fallen short of their initial promise and hype. The observed marginal, to no benefit, success in several applications has been attributed primarily to poor cell survival and engraftment at transplantation sites. MSCs have a metabolism that is flexible enough to enable them to fulfill their various cellular functions and remarkably sensitive to different cellular and environmental cues. At the transplantation sites, MSCs experience hostile environments devoid or, at the very least, severely depleted of oxygen and nutrients. The impact of this particular setting on MSC metabolism ultimately affects their survival and function. In order to develop the next generation of cell-delivery materials and methods, scientists must have a better understanding of the metabolic switches MSCs experience upon transplantation. By designing treatment strategies with cell metabolism in mind, scientists may improve survival and the overall therapeutic potential of MSCs. Here, we provide a comprehensive review of plausible metabolic switches in response to implantation and of the various strategies currently used to leverage MSC metabolism to improve stem cell-based therapeutics.

Using notochordal cells of developmental origin to stimulate nucleus pulposus cells and bone marrow stromal cells for intervertebral disc regeneration.

PURPOSE: Bone marrow stromal cells (BMSCs) have been proposed to complement the declining population of nucleus pulposus cells (NPCs) found in a degenerative intervertebral disc. Although able to stop degeneration, they could not produce enough matrix to restore a healthy state. Looking at development, when a large amount of matrix is produced, the disc also contains notochordal cells (NCs), potential progenitors or regulators of NPCs. The aim of the study was, therefore, to combine NCs to a BMSC/NPC mix and evaluate their effects on cell phenotype and matrix production, in long-term culture. METHODS: In a 3D hydrogel, NCs were co-cultured in different ratios with BMSCs and/or NPCs. Matrix production, cell morphology, and gene expression of disc markers were assessed after 4 weeks of culture. RESULTS: At day 28, BMSCs/NPCs highly expressed disc matrix markers (type II collagen and aggrecan) and produced disc matrix up to 30 % of values obtained for the positive control (BMSCs under TGFß stimulation). The addition of NCs only slightly up-regulated marker expression (6-12× increase); an up-regulation not reflected at the matrix level. During the 4 weeks of culture, however, the NC phenotype changed drastically (morphology, disc marker expression). CONCLUSION: In contrast to previously reported short-term studies, long-term co-cultures with NCs had no substantial effects on BMSCs and NPCs, most likely due to the loss of the NC native phenotype during culture. It, therefore, appears critical to maintain this specific phenotype for a long-term effect of the NCs.

Porosity and surface curvature effects on the permeability and wall shear stress of trabecular bone: Guidelines for biomimetic scaffolds for bone repair.

Scaffolds are an essential component of bone tissue engineering to provide support and create a physiological environment for cells. Biomimetic scaffolds are a promising approach to fulfill the requirements. Bone allografts are widely used scaffolds due to their mechanical and structural characteristics. The scaffold geometry is well known to be an important determinant of induced mechanical stimulation felt by the cells. However, the impact of allograft geometry on permeability and wall shear stress distribution is not well understood. This information is essential for designing biomimetic scaffolds that provide a suitable environment for cells to proliferate and differentiate. The present study investigates the effect of geometry on the permeability and wall shear stress of bone allografts at both macroscopic and microscopic scales. Our results concluded that the wall shear stress was strongly correlated with the porosity of the allograft. The level of wall shear stress at a local scale was also determined by the surface curvature characteristics. The results of this study can serve as a guideline for future biomimetic scaffold designs that provide a mechanical environment favorable for osteogenesis and bone repair.

Glucose depletion decreases cell viability without triggering degenerative changes in a physiological nucleus pulposus explant model.

Although the etiology of intervertebral disc degeneration is still unresolved, the nutrient paucity resulting from its avascular nature is suspected of triggering degenerative processes in its core: the nucleus pulposus (NP). While severe hypoxia has no significant effects on NP cells, the impact of glucose depletion, such as found in degenerated discs (0.2-1 mM), is still uncertain. Using a pertinent ex-vivo model representative of the unique disc microenvironment, the present study aimed, therefore, at determining the effects of "degenerated" (0.3 mM) glucose levels on bovine NP explant homeostasis. The effects of glucose depletion were evaluated on NP cell viability, apoptosis, phenotype, metabolism, senescence, extracellular matrix anabolism and catabolism, and inflammatory mediator production using fluorescent staining, RT-qPCR, (immuno)histology, ELISA, biochemical, and enzymatic assays. Compared to the "healthy" (2 mM) glucose condition, exposure to the degenerated glucose condition led to a rapid and extensive decrease in NP cell viability associated with increased apoptosis. Although the aggrecan and collagen-II gene expression was also downregulated, NP cell phenotype, and senescence, matrix catabolism, and inflammatory mediator production were not, or only slightly, affected by glucose depletion. The present study provided evidence for glucose depletion as an essential player in NP cell viability but also suggested that other microenvironment factor(s) may be involved in triggering the typical shift of NP cell phenotype observed during disc degeneration. The present study contributes new information for better understanding disc degeneration at the cellular-molecular levels and thus helps to develop relevant therapeutical strategies to counteract it.

The Lower in Vivo Osteogenicity of Adipose Tissue-Derived Stem Cells Correlates with a Higher Innate Immune Response.

Adipose tissue-derived mesenchymal stem cells (ATSCs) have been used as an alternative to bone marrow-derived mesenchymal stem cells (BMSCs) for bone tissue engineering applications. The ability of ATSCs to promote new bone formation remains lower than that of BMSCs. This study aimed to investigate the mechanisms underlying osteogenicity differences between human ATSCs and BMSCs in ceramic constructs, focusing on the effects of inflammation on this process. In contrast to ATSC-containing constructs, which did not induce bone formation in an ectopic mouse model, BMSC constructs consistently did so. Gene expression analysis revealed that human BMSCs, concomitantly with host murine progenitors, differentiated into the osteogenic lineage early post-implantation. In contrast, ATSCs differentiated later, when few implanted viable cells remained post-implantation, while the host murine cells did not differentiate. Comparison of the inflammatory profile in the cell constructs indicated concomitant upregulation of some human and murine inflammatory genes in the ATSC-constructs compared to the BMSC-constructs during the first-week post-implantation. The high level of chemokine production by the ATSCs was confirmed at the gene and protein levels before implantation. The immune cell recruitment within the constructs was then explored post-implantation. Higher numbers of TRAP-/ MRC1 (CD206) + multinucleated giant cells, NOS2 + M1, and ARG1 + M2 macrophages were present in the ATSC constructs than in the BMSC constructs. These results proved that ATSCs are a transient source of inflammatory cytokines promoting a transient immune response post-implantation; this milieu correlates with impaired osteogenic differentiation of both the implanted ATSCs and the host osteoprogenitor cells.

A New Microarchitecture-Based Parameter to Predict the Micromechanical Properties of Bone Allografts.

Scaffolds are an essential component of bone tissue engineering. They provide support and create a physiological environment for cells to proliferate and differentiate. Bone allografts extracted from human donors are promising scaffolds due to their mechanical and structural characteristics. Bone microarchitecture is well known to be an important determinant of macroscopic mechanical properties, but its role at the microscopic, i.e., the trabeculae level is still poorly understood. The present study investigated linear correlations between microarchitectural parameters obtained from X-ray computed tomography (micro-CT) images of bone allografts, such as bone volume fraction (BV/TV), degree of anisotropy (DA), or ellipsoid factor (EF), and micromechanical parameters derived from micro-finite element calculations, such as mean axial strain (εz) and strain energy density (We). DAEF, a new parameter based on a linear combination of the two microarchitectural parameters DA and EF, showed a strong linear correlation with the bone mechanical characteristics at the microscopic scale. Our results concluded that the spatial distribution and the plate-and-rod structure of trabecular bone are the main determinants of the mechanical properties of bone at the microscopic level. The DAEF parameter could, therefore, be used as a tool to predict the level of mechanical stimulation at the local scale, a key parameter to better understand and optimize the mechanism of osteogenesis in bone tissue engineering.

Glucose Metabolism: Optimizing Regenerative Functionalities of Mesenchymal Stromal Cells Postimplantation.

Mesenchymal stromal cells (MSCs) are considered promising candidates for regenerative medicine applications. Their clinical performance postimplantation, however, has been disappointing. This lack of therapeutic efficacy is most likely due to suboptimal formulations of MSC-containing material constructs. Tissue engineers, therefore, have developed strategies addressing/incorporating optimized cell, microenvironmental, biochemical, and biophysical cues/stimuli to enhance MSC-containing construct performance. Such approaches have had limited success because they overlooked that maintenance of MSC viability after implantation for a sufficient time is necessary for MSCs to develop their regenerative functionalities fully. Following a brief overview of glucose metabolism and regulation in MSCs, the present literature review includes recent pertinent findings that challenge old paradigms and notions. We hereby report that glucose is the primary energy substrate for MSCs, provides precursors for biomass generation, and regulates MSC functions, including proliferation and immunosuppressive properties. More importantly, glucose metabolism is central in controlling in vitro MSC expansion, in vivo MSC viability, and MSC-mediated angiogenesis postimplantation when addressing MSC-based therapies. Meanwhile, in silico models are highlighted for predicting the glucose needs of MSCs in specific regenerative medicine settings, which will eventually enable tissue engineers to design viable and potent tissue constructs. This new knowledge should be incorporated into developing novel effective MSC-based therapies. Impact statement The clinical use of mesenchymal stromal cells (MSCs) has been unsatisfactory due to the inability of MSCs to survive and be functional after implantation for sufficient periods to mediate directly or indirectly a successful regenerative tissue response. The present review summarizes the endeavors in the past, but, most importantly, reports the latest findings that elucidate underlying mechanisms and identify glucose metabolism as the crucial parameter in MSC survival and the subsequent functions pertinent to new tissue formation of importance in tissue regeneration applications. These latest findings justify further basic research and the impetus for developing new strategies to improve the modalities and efficacy of MSC-based therapies.

Combining biomimetic collagen/hyaluronan hydrogels with discogenic growth factors promotes mesenchymal stroma cell differentiation into Nucleus Pulposus like cells.

Based on stem cell injection into degenerated Nucleus Pulposus (NP), novel treatments for intervertebral disc (IVD) regeneration were disappointing because of cell leakage or inappropriate cell differentiation. In this study, we hypothesized that mesenchymal stromal cells encapsulated within injectable hydrogels possessing adequate physico-chemical properties would differentiate into NP like cells. Composite hydrogels consisting of type I collagen and tyramine-substituted hyaluronic acid (THA) were prepared to mimic the NP physico-chemical properties. Human bone marrow derived mesenchymal stromal cells (BM-MSCs) were encapsulated within hydrogels and cultivated in proliferation medium (supplemented with 10% fetal bovine serum) or differentiation medium (supplemented with GDF5 and TGFß1) over 28 days. Unlike pure collagen, collagen/THA composite hydrogels were stable over 28 days in culture. In proliferation medium, the cell viability within pure collagen hydrogels was high, whereas that in composite and pure THA hydrogels was lower due to the weaker cell adhesion. Nonetheless, BM-MSCs proliferated in all hydrogels. In composite hydrogels, cells exhibited a rounded morphology similar to NP cells. The differentiation medium did not impact the hydrogel stability and cell morphology but negatively impacted the cell viability in pure collagen hydrogels. A high THA content within hydrogels promoted the gene expression of NP markers such as collagen II, aggrecan, SOX9 and cytokeratin 18 at day 28. The differentiation medium potentialized this effect with an earlier and higher expression of these NP markers. Taken together, these results show that the physico-chemical properties of collagen/THA composite hydrogels and GDF5/TGFß1 act in synergy to promote the differentiation of BM-MSCs into NP like cells.

Enzyme-controlled, nutritive hydrogel for mesenchymal stromal cell survival and paracrine functions.

Culture-adapted human mesenchymal stromal cells (hMSCs) are appealing candidates for regenerative medicine applications. However, these cells implanted in lesions as single cells or tissue constructs encounter an ischemic microenvironment responsible for their massive death post-transplantation, a major roadblock to successful clinical therapies. We hereby propose a paradigm shift for enhancing hMSC survival by designing, developing, and testing an enzyme-controlled, nutritive hydrogel with an inbuilt glucose delivery system for the first time. This hydrogel, composed of fibrin, starch (a polymer of glucose), and amyloglucosidase (AMG, an enzyme that hydrolyze glucose from starch), provides physiological glucose levels to fuel hMSCs via glycolysis. hMSCs loaded in these hydrogels and exposed to near anoxia (0.1% pO2) in vitro exhibited improved cell viability and angioinductive functions for up to 14 days. Most importantly, these nutritive hydrogels promoted hMSC viability and paracrine functions when implanted ectopically. Our findings suggest that local glucose delivery via the proposed nutritive hydrogel can be an efficient approach to improve hMSC-based therapeutic efficacy.

Three-dimensional Printing of Biomimetic Titanium Mimicking Trabecular Bone Induces Human Mesenchymal Stem Cell Proliferation: An In-vitro Analysis.

STUDY DESIGN: In vitro analysis. OBJECTIVE: The aim of this study was to assess the effect of three-dimensional (3D) printing of porous titanium on human mesenchymal stem cell (hMSC) adhesion, proliferation, and osteogenic differentiation. SUMMARY OF BACKGROUND DATA: A proprietary implant using three-dimensional porous titanium (3D-pTi) that mimics trabecu-lar bone structure, roughness, porosity, and modulus of elasticity was created (Ti-LIFE technology™, Spineart SA Switzerland). Such implants may possess osteoinductive properties augmenting fusion in addition to their structural advantages. However, the ability of 3D-pTi to affect in vitro cellular proliferation and osteogenic differentiation remains undefined. METHODS: Disks of 3D-pTi with a porosity of 70% to 75% and pore size of 0.9 mm were produced using additive manufacturing technology. 2D Ti6Al4V (2D-Ti) and 2D polyetheretherketone (2D-PEEK) disks were prepared using standard manufacturing process. Tissue culture plastic (TCP) served as the control surface. All discs were characterized using 2D-micros-copy, scanning electron microscopy (SEM), and x-ray micro-computed tomography. Forty thousand hMSCs were seeded on the disks and TCP and cultured for 42 days. hMSC morphology was assessed using environmental SEM and confocal imaging following phalloidin staining. hMSC proliferation was evaluated using DNA fluorescent assay. hMSC differentiation was assessed using RT-qPCR for genes involved in hMSC osteogenic differentiation and biochemical assays were performed for alkaline phosphatase activity (ALP) and calcium content. RESULTS: 3D-pTi lead to a higher cell number as compared to 2D-Ti and 2D-PEEK at D21, D28 and D42. ALP activity of hMSCs seeded into 3D-pTi scaffolds was as high as or higher than that of hMSCs seeded onto TCP controls over all time points and consistently higher than that of hMSCs seeded onto 2D-Ti scaffolds. However, when ALP activity was normalized to protein content, no statistical differences were found between all scaffolds tested and TCP controls. CONCLUSION: 3D-pTi provides a scaffold for bone formation that structurally mimics cancellous bone and improves hMSC adhesion and proliferation compared to 2D-Ti and PEEK.

Osteogenic-differentiated mesenchymal stem cell-secreted extracellular matrix as a bone morphogenetic protein-2 delivery system for ectopic bone formation.

While human bone morphogenetic protein-2 (BMP-2) is a promising growth factor for bone regeneration, a major challenge in biomedical applications is finding an optimal carrier for its delivery at the site of injury. Because of their natural affinities for growth factors (including BMP-2) as well as their role in instructing cell function, cultured cell-derived extracellular matrices (ECM) are of special interest. We hereby hypothesized that a "bony matrix" containing mineralized, osteogenic ECM is a potential efficacious carrier of BMP-2 for promoting bone formation and, therefore, compared the efficacy of the decellularized ECM derived from osteogenic-differentiated human mesenchymal stem cells (hMSCs) to the one obtained from ECM from undifferentiated hMSCs. Our results provided evidence that both ECMs can bind BMP-2 and promote bone formation when implanted ectopically in mice. The osteoinductive potential of BMP-2, however, was greater when loaded within an osteogenic MSC-derived ECM; this outcome was correlated with higher sequestration capacity of BMP-2 over time in vivo. Interestingly, although the BMP-2 mainly bound onto the mineral crystals contained within the osteogenic MSC derived-ECM, these mineral components were not involved in the observed higher osteoinductivity, suggesting that the organic components were the critical components for the matrix efficacy as BMP-2 carrier.

Custom-made macroporous bioceramic implants based on triply-periodic minimal surfaces for bone defects in load-bearing sites.

The architectural features of synthetic bone grafts are key parameters for regulating cell functions and tissue formation for the successful repair of bone defects. In this regard, macroporous structures based on triply-periodic minimal surfaces (TPMS) are considered to have untapped potential. In the present study, custom-made implants based on a gyroid structure, with (GPRC) and without (GP) a cortical-like reinforcement, were specifically designed to fit an intended bone defect in rat femurs. Sintered hydroxyapatite implants were produced using a dedicated additive manufacturing technology and their morphological, physico-chemical and mechanical features were characterized. The implants' integrity and ability to support bone ingrowth were assessed after 4, 6 and 8 weeks of implantation in a 3-mm-long, femoral defect in Lewis rats. GP and GPRC implants were manufactured with comparable macro- to nano-architectures. Cortical-like reinforcement significantly improved implant effective stiffness and resistance to fracture after implantation. This cortical-like reinforcement also concentrated new bone formation in the core of the GPRC implants, without affecting newly formed bone quantity or maturity. This study showed, for the first time, that custom-made TPMS-based bioceramic implants could be produced and successfully implanted in load-bearing sites. Adding a cortical-like reinforcement (GPRC implants) was a relevant solution to improve implant mechanical resistance, and changed osteogenic mechanism compared to the GP implants. STATEMENT OF SIGNIFICANCE: Architectural features are known to be key parameters for successful bone repair using synthetic bioceramic bone graft. So far, conventional manufacturing techniques, lacking reproducibility and complete control of the implant macro-architecture, impeded the exploration of complex architectures, such as triply periodic minimal surfaces (TPMS), which are foreseen to have an unrivaled potential for bone repair. Using a new additive manufacturing process, macroporous TPMS-based bioceramics implants were produced in calcium phosphate, characterized and implanted in a femoral defect in rats. The results showed, for the first time, that such macroporous implants can be successfully implanted in anatomical load-bearing sites when a cortical-like outer shell is added. This outer shell also concentrated new bone formation in the implant center, without affecting new bone quantity or maturity.

Osteogenic potential of adipogenic predifferentiated human bone marrow-derived multipotent stromal cells for bone tissue-engineering.

In the present study, we evaluated the benefits of an adipogenic predifferentiation, the pathway most closely related to osteoblastogenesis, on the pro-osteogenic potential of human adult multipotent bone marrow stromal cells (hBMSCs), both in vitro and in vivo. Adipogenic differentiation of hBMSCs for 14 days resulted in a heterogeneous cell population from which the most adipogenic-committed cells were eliminated by their lack of readhesion ability. Our results provided evidence that the select adherent adipogenic differentiated hBMSCs (sAD+ cells) express a gene profile characteristic of both adipogenic and osteogenic lineages. In vitro, when cultured in osteogenic medium, sAD+ differentiated along the osteogenic lineage faster than undifferentiated hBMSCs. In vivo, in an ectopic mouse model, sAD+ exhibited a significantly higher bone formation capability compared with undifferentiated hBMSCs. We sought, then, to investigate the underlying mechanisms responsible for such beneficial effects of adipogenic predifferentiation on bone formation and found that this outcome was not linked to a better cell survival post-implantation. The secretome of sAD+ was both proangiogenic and chemoattractant, but its potential did not supersede the one of undifferentiated hBMSCs. However, using co-culture systems, we observed that the sAD+ paracrine factors were pro-osteogenic on undifferentiated hBMSCs. In conclusion, adipogenic priming endows hBMSCs with high osteogenic potential as well as pro-osteogenic paracrine-mediated activity. This preconditioning appears as a promising strategy for bone tissue engineering technology in order to improve the hBMSC osteogenic potency in vivo.

Hypoxia affects mesenchymal stromal cell osteogenic differentiation and angiogenic factor expression.

Mesenchymal stromal cells (MSCs) seeded onto biocompatible scaffolds have been proposed for repairing bone defects. When transplanted in vivo, MSCs (expanded in vitro in 21% O(2)) undergo temporary oxygen deprivation due to the lack of pre-existing blood vessels within these scaffolds. In the present study, the effects of temporary (48 h) exposure to hypoxia (

Prolonged hypoxia concomitant with serum deprivation induces massive human mesenchymal stem cell death.

Mesenchymal stem cells (MSCs) have been proposed for the repair of damaged tissue including bone, cartilage, and heart tissue. Upon in vivo transplantation, the MSCs encounter an ischemic microenvironment characterized by reduced oxygen (O2) tension and nutrient deprivation that may jeopardize viability of the tissue construct. The aim of this study was to assess the effects of serum deprivation and hypoxia on the MSC survival rates in vitro. As expanded MSCs are transferred from plastic to a scaffold in most tissue engineering approaches, possibly inducing loss of survival signals from matrix attachments, the effects of a scaffold shift on the MSC survival rates were also assessed. Human MSCs were exposed for 48 hours to (i) a scaffold substrate shift, (ii) serum deprivation, and (iii) O2 deprivation. MSCs were also exposed to prolonged (up to 120 hours) hypoxia associated with serum deprivation. Cell death was assessed by Live/Dead staining and image analysis. The MSC death rates were not affected by the shift to scaffold or 48-hour hypoxia, but increased with fetal bovine serum (FBS) starvation, suggesting that between the two components of ischemia, nutrient deprivation is the stronger factor. Long-term hypoxia combined with serum deprivation resulted in the complete death of MSCs (99 +/- 1%), but this rate was reduced by half when MSCs were exposed to hypoxia in the presence of 10% FBS (51 +/- 31%). These results show that MSCs are sensitive to the concurrent serum and O2 deprivation to which they are exposed when transplanted in vivo, and call for the development of new transplantation methods.

The Regenerative Potential of Notochordal Cells in a Nucleus Pulposus Explant.

STUDY DESIGN: In vitro disk explant culture. OBJECTIVE: Notochordal cells (NCs) have been shown to upregulate matrix production by nucleus pulposus (NP) cells in coculture. To examine the translation of these in vitro results to a nativelike setting, the regenerative potential of NCs injected into NP tissue was assessed in this study. METHODS: NP explants were cultured after injection with NCs in phosphate-buffered saline (PBS) or with PBS alone (sham). At days 0 and 42, cell viability and morphology, water, DNA, sulfated glycosaminoglycan and hydroxyproline content, and gene expression of anabolic markers were analyzed. RESULTS: NCs remained viable during culture, but their morphology changed. The biochemical content remained unchanged, except for the DNA content in the NC group. Overall ACAN expression remained unchanged, whereas COL2A1 decreased during culture. CONCLUSIONS: No overall anabolic response was observed when NCs were injected into NP explants. NCs were found to survive but did not display the typical NC morphology by the end of the culture period.

Osteogenic protein 1 does not stimulate a regenerative effect in cultured human degenerated nucleus pulposus tissue.

Low back pain is a major cause of disability and is heavily associated with intervertebral disc degeneration. Osteogenic protein 1 (OP-1) is a growth factor that has shown potential to regenerate the intervertebral disc in human cells and animal models. However, high doses are required, presumably due to clearance from the tissue; controlled release may be a solution to this problem. In this study, we developed a preclinical, pathophysiological human tissue explant culture model of degenerated nucleus pulposus (NP). The NP explants were cultured for 28 days and injected with 100 µg OP-1 as a bolus, or with sustained-release biodegradable microspheres loaded with 16 or 1.6 µg OP-1. After culture, the tissue explants were analysed for biochemical content [water, sulphated glycosaminoglycans (GAGs), hydroxyproline and DNA], histology, cell viability and gene expression (disc matrix anabolic and catabolic markers). Untreated degenerated NP explants lost some of their GAG content when cultured for 4 weeks, but maintained other tissue constituents. Gene expression levels were close to native values. A bolus injection of OP-1 partially restored GAG content to the native level in half of the donors, while the sustained release of OP-1 did not affect the NP explants. No effect of treatment was observed on anabolic or catabolic gene expression at day 28. These results demonstrated that the regenerative potential of OP-1 is donor dependent, and only at very high doses. This questions the clinical use of OP-1 as a regenerative agent, as these high doses may increase the incidence of complications. Copyright © 2015 John Wiley & Sons, Ltd.

Reduced tonicity stimulates an inflammatory response in nucleus pulposus tissue that can be limited by a COX-2-specific inhibitor.

In intervertebral disc herniation with nucleus pulposus (NP) extrusion, the elicited inflammatory response is considered a key pain mechanism. However, inflammatory cytokines are reported in extruded herniated tissue, even before monocyte infiltration, suggesting that the tissue itself initiates the inflammation. Since herniated tissue swells, we investigated whether this simple mechanobiological stimulus alone could provoke an inflammatory response that could cause pain. Furthermore, we investigated whether sustained-release cyclooxygenase-2 (COX2) inhibitor would be beneficial in such conditions. Healthy bovine NP explants were allowed to swell freely or confined. The swelling explants were treated with Celecoxib, applied either as a bolus or in sustained-release. Swelling explants produced elevated levels of interleukin-6 (IL-6) and prostaglandin E2 (PGE2 ) for 28 days, while confined explants did not. Both a high concentration bolus and 10 times lower concentration in sustained release completely inhibited PGE2 production, but did not affect IL-6 production. Swelling of NP tissue, without the inflammatory system response, can trigger cytokine production and Celecoxib, even in bolus form, may be useful for pain control in extruded disc herniation.

Conditioned medium derived from notochordal cell-rich nucleus pulposus tissue stimulates matrix production by canine nucleus pulposus cells and bone marrow-derived stromal cells.

OBJECTIVES: Conditioned medium derived from notochordal cell-rich nucleus pulposus tissue (NCCM) was previously shown to have a stimulatory effect on bone marrow stromal cells (BMSCs) and nucleus pulposus cells (NPCs) individually, in mixed species in vitro cell models. The objective of the current study was to assess the stimulatory effect of NCCM on NPCs in a homologous canine in vitro model and to investigate whether combined stimulation with NCCM and addition of BMSCs provides a synergistic stimulatory effect. METHODS: BMSCs and NPCs were harvested from chondrodystrophic dogs with confirmed early intervertebral disc (IVD) degeneration. NCCM was produced from NP tissue of nonchondrodystrophic dogs with healthy IVDs. BMSCs or NPCs alone (3×10(6) cells/mL) and NPCs+BMSCs (6×10(6) cells/mL; mixed 1:1) were cultured for 4 weeks in 1.2% alginate beads under base medium (BM), NCCM, or with addition of 10 ng/mL transforming growth factor-ß1 (TGF-ß1) as a positive control. Beads were assessed for glycosaminoglycan (GAG) and DNA contents by biochemical assays, GAG deposition by Alcian blue staining, and gene expression (aggrecan, versican, collagen 1 and 2, SOX9, A disintegrin and metalloproteinase with thrombospondin motifs 5 (ADAMTS5), and matrix metalloproteinase 13 [MMP13]) with real-time quantitative RT-PCR. RESULTS: NCCM increased NPC proliferation, proteoglycan production, and expression of genes associated with a healthy NP-like phenotype. BMSCs also showed increased proteoglycan production under NCCM, but these effects were not observed at the gene level. Combined stimulation of NPCs with NCCM and coculturing with BMSCs did not result in increased proteoglycan content compared to stimulation with NCCM alone. DISCUSSION: NCCM stimulates matrix production by both NPCs and BMSCs and directs NPCs toward a healthier phenotype. NCCM is therefore promising for IVD regeneration and identification of the bioactive components will be helpful to further develop this approach. In the current study, no synergistic effect of adding BMSCs was observed.

Effect of coculturing canine notochordal, nucleus pulposus and mesenchymal stromal cells for intervertebral disc regeneration.

INTRODUCTION: Early degenerative changes in the nucleus pulposus (NP) are observed after the disappearance of notochordal cells (NCs). Thus, it has been suggested that NCs play an important role in maintaining the NP and may have a regenerative potential on other cells of the NP. As the number of resident NP cells (NPCs) decreases in a degenerating disc, mesenchymal stromal (stem) cells (MSCs) may be used for cell supplementation. In this study, using cells of one species, the regenerative potential of canine NCs was assessed in long-term three-dimensional coculture with canine NPCs or MSCs. METHODS: Canine NCs and canine NPCs or MSCs were cocultured in alginate beads for 28 days under hypoxic and high-osmolarity conditions. Cell viability, cell morphology and DNA content, extracellular matrix production and expression of genes related to NC markers (Brachyury, KRT18) and NP matrix production (ACAN, COL2A1, COL1A1) were assessed after 1, 15 and 28 days of culture. RESULTS: NCs did not completely maintain their phenotype (morphology, matrix production, gene expression) during 28 days of culture. In cocultures of NPCs and NCs, both extracellular matrix content and anabolic gene expression remained unchanged compared with monoculture groups, whereas cocultures of MSCs and NCs showed increased glycosaminoglycan/DNA. However, the deposition of these proteoglycans was observed near the NCs and not the MSCs. Brachyury expression in the MSC and NC coculture group increased in time. The latter two findings indicate a trophic effect of MSCs on NCs rather than vice versa. CONCLUSIONS: No regenerative potential of canine NCs on canine NPCs or MSCs was observed in this study. However, significant changes in NC phenotype in long-term culture may have resulted in a suboptimal regenerative potential of these NCs. In this respect, NC-conditioned medium may be better than coculture for future studies of the regenerative potential of NCs.