Hepatocyte growth factor/c‐met promotes proliferation,suppresses apoptosis,and improves matrix metabolism in rabbit nucleus pulposus cells in vitro

The etiology of intervertebral disc (IVD) degeneration is closely related to apoptosis and extracellular matrix degradation in nucleus pulposus (NP) cells. These defects in NP cells are induced by excessive external stressors such as reactive oxygen species (ROS) and inflammatory cytokines. Recently, hepatocyte growth factor (HGF) has been shown to repair damage in various diseases through anti‐apoptotic and anti‐inflammatory activity. In this study, we investigated the effects of HGF on NP cell abnormality caused by ROS and inflammatory cytokines by using primary NP cells isolated from rabbit IVD. HGF significantly enhanced the proliferation of NP cells. Apoptosis of NP cells induced by H₂O₂ or TNF‐α was significantly inhibited by HGF. Induction of mRNA expression of the inflammation mediators cyclooxygenase‐2 and matrix metalloproteinase‐3 and ‐9 by TNF‐α was significantly suppressed by HGF treatment. Expression of c‐Met, a specific receptor for HGF, was confirmed in NP cells and was increased by TNF‐α, suggesting that inflammatory cytokines increase sensitivity to HGF. These findings demonstrate that activation of HGF/c‐Met signaling suppresses damage caused by ROS and inflammation in NP cells through multiple pathways. We further suggest the clinical potential of HGF for counteracting IVD degradation involved in NP cell abnormalities. © 2015 Orthopaedic Research Society. Published by Wiley Periodicals, Inc. J Orthop Res 34:709–716, 2016.     

Role of SHOX2 in the development of intervertebral disc degeneration

Intervertebral disc (IVD) degeneration is the most common cause of low back pain, which affect 80% of the population during their lives, with heavy economic burden. Many factors have been demonstrated to participate in IVD degeneration. In this study, we investigated the role of short stature homeobox 2 (SHOX2) in the development of IVD degeneration. First, we detected the expression of SHOX2 in different stages of human IVD degeneration; then explored the role of SHOX2 on nucleus pulposus (NP) cells proliferation and apoptosis, finally we evaluated the effect of SHOX2 on the production of extracellular matrix in NP cells. Results showed that the expression of SHOX2 is mainly in NP compared with AF tissues, its expression decreased with the severity of human IVD degeneration. TNF‐α treatment led to dose‐ and time‐dependent decrease in SHOX2 mRNA, protein expression and promoter activity in NP cells. The silencing of SHOX2 inhibited NP cells proliferation and induced NP cells apoptosis. Finally, SHOX2 silencing led to decreased aggrecan and collagen II expression, along with increased ECM degrading enzymes MMP3 and ADAMTS‐5 in NP cells. In summary, our results indicated that SHOX2 plays an important role in the process of IVD degeneration, and might be a protective factor for IVD degeneration. Further studies are required to confirm its exact role, and clarify the mechanism. © 2017 Orthopaedic Research Society. Published by Wiley Periodicals, Inc. J Orthop Res 35:1047–1057, 2017.

     

Energy metabolism of intervertebral disc under mechanical loading

Intervertebral disc (IVD) degeneration is closely associated with low back pain (LBP), which is a major health concern in the U.S. Cellular biosynthesis of extracellular matrix (ECM), which is important for maintaining tissue integrity and preventing tissue degeneration, is an energy demanding process. Due to impaired nutrient support in avascular IVD, adenosine triphosphate (ATP) supply could be a limiting factor for maintaining normal ECM synthesis. Therefore, the objective of this study was to investigate the energy metabolism in the annulus fibrosus (AF) and nucleus pulposus (NP) of porcine IVD under static and dynamic compressions. Under compression, pH decreased and the contents of lactate and ATP increased significantly in both AF and NP regions, suggesting that compression can promote ATP production via glycolysis and reduce pH by increasing lactate accumulation. A high level of extracellular ATP content was detected in the NP region and regulated by compressive loading. Since ATP can serve not only as an intra‐cellular energy currency, but also as a regulator of a variety of cellular activities extracellularly through the purinergic signaling pathway, our findings suggest that compression‐mediated ATP metabolism could be a novel mechanobiological pathway for regulating IVD metabolism. © 2013 Orthopaedic Research Society. Published by Wiley Periodicals, Inc. J Orthop Res 31:1733–1738, 2013     

Induction of matrix metalloproteinase-2 and -3 activity in ovine nucleus pulposus cells grown in three-dimensional agarose gel culture by interleukin-1β: a potential pathway of disc degeneration

Degeneration of the intervertebral disc is an important clinical problem, which often contributes to low back pain. Since approximately 80-90% of the general population will be subject to back pain at some stage during their lifetime, this has major socioeconomic consequences. Matrix metalloproteinases (MMPs) have been implicated in the excessive breakdown of extracellular matrix components during disc degeneration. The aim of the present study was to evaluate the regulation of MMP-2 (gelatinase-A) and MMP-3 (stromelysin) produced by cultured ovine nucleus pulposus (NP) cells stimulated with interleukin-1beta (IL-1beta). NP cells were established in three-dimensional agarose culture and stimulated with IL-1beta under serum-free conditions. Conditioned media samples were evaluated by gelatin and casein zymography and by fluorimetry using an MMP-specific substrate. Time-course and dose dependencies were established for MMP-2, -3 production by the NP cells in response to the IL-1beta. Gelatin and casein zymography indicated that elevated levels of proMMP-2 and proMMP-3 were present in media samples in response to the IL-1beta treatment. After 24-96 h culture, levels of the active 43 and 45 kDa active MMP-3 were significantly elevated, whereas MMP-2 was present mainly as its 72 kDa pro-form. Additional 36, 28 and 21 kDa MMP species were also present after prolonged incubation with IL-1beta, probably representing MMP breakdown species. IL-1beta was a potent catabolic mediator for the NP cells, resulting in the production of elevated levels of MMP-2 and -3 in culture. However, approximately 70% of the MMP-2 was present as the 72 kDa pro-form, which suggests that some additional steps are involved in its activation in vivo.     

Impact of direct cell co‐cultures on human adipose‐derived stromal cells and nucleus pulposus cells

Biologic and cellular treatment strategies aiming for curing intervertebral disc degeneration (IDD) have been proposed recently. Given the convenient availability and expansion potential, adipose‐derived stromal cells (ADSCs) might be an ideal cell candidate. However, the interaction between ADSCs and nucleus pulposus (NP) cells still remains ambiguous, especially in direct co‐cultures of the two types of cells. Nevertheless, NP markers in ADSCs after co‐cultures were unidentified. Here, we addressed the interaction of human ADSCs and NP cells in a direct co‐culture system for the first time. As a result, ADSCs could differentiate to the NP cell phenotype with a significant up‐regulated expression of multiple genes and proteins in extracellular matrix (ECM) (SOX9, COL2A1, ACAN, and COL6A2), relative NP markers (FOXF1, PAX1, CA12, and HBB) and pertinent growth factors (CDMP‐1, TGF‐β1, IGF‐1, and CTGF). Moreover, the gene expression of COL2A1, ACAN, and COL6A2 of degenerate NP cells was also up‐regulated. Collectively, these results suggest that direct co‐cultures of ADSCs and NP cells may exert a reciprocal impact, that is, both stimulating ADSCs differentiation to the NP cell phenotype and inducing NP cells to regain functional phenotype. Accordingly, ADSCs might be a potential candidate in the development of cellular treatment strategies for IDD. © 2013 Orthopaedic Research Society. Published by Wiley Periodicals, Inc. J Orthop Res 31:1804–1813, 2013     

Long-term culture of bovine nucleus pulposus explants in a native environment

Background contextChronic low back pain is a disease with tremendous financial and social implications, and it is often caused by intervertebral disc degeneration. Regenerative therapies for disc repair are promising treatments, but they need to be tested in physiological models.PurposeTo develop a physiological in vitro explant model that incorporates the native environment of the intervertebral disc, for example, hypoxia, low glucose, and high tissue osmolarity.Study designBovine nucleus pulposus (NP) explants were cultured for 42 days in conditions mimicking the native physiological environment. Two different approaches were used to balance the swelling pressure of the NP: raised medium osmolarity or an artificial annulus.MethodsBovine NP explants were either cultured in media with osmolarity balanced at isotonic and hypertonic levels compared with the native tissue or cultured inside a fiber jacket used as an artificial annulus. Oxygen and glucose levels were set at either standard (21% O₂ and 4.5 g/L glucose) or physiological (5% O₂ and 1 g/L glucose) levels. Samples were analyzed at Day 0, 3, and 42 for tissue composition (water, sulfated glycosaminoglycans, DNA, and hydroxyproline contents and fixed charge density), tissue histology, cell viability, and cellular behavior with messenger RNA (mRNA) expression.ResultsBoth the hypertonic culture and the artificial annulus approach maintained the tissue matrix composition for 42 days. At Day 3, mRNA expressions of aggrecan, collagen Type I, and collagen Type II in both hypertonic and artificial annulus cultures were not different from Day 0; however, at Day 42, the artificial annulus preserved the mRNA expression closer to Day 0. Gene expressions of matrix metalloprotease 13, tissue inhibitor of matrix metalloprotease 1, and tissue inhibitor of matrix metalloprotease 2 were downregulated under physiological O₂ and glucose levels, whereas the other parameters analyzed were not affected.ConclusionsAlthough the hypertonic culture and the artificial annulus approach are both promising models to test regenerative therapies, the artificial annulus was better able to maintain a cellular behavior closer to the native tissue in longer term cultures.     

Osmolarity affects matrix synthesis in the nucleus pulposus associated with the involvement of MAPK pathways: A study of ex vivo disc organ culture system

Matrix homeostasis within the nucleus pulposus (NP) is important for disc function. Unfortunately, the effects of osmolarity on NP matrix synthesis in a disc organ culture system and the underlying mechanisms are largely unknown. The present study was to investigate the effects of different osmolarity modes (constant and cyclic) and osmolarity levels (hypo‐, iso‐, and hyper‐) on NP matrix synthesis using a disc organ culture system and determine whether ERK1/2 or p38MAPK pathway has a role in this process. Porcine discs were cultured for 7 days in various osmotic media, including constant hypo‐, iso‐, hyper‐osmolarity (330, 430, and 550 mOsm/kg, respectively) and cyclic‐osmolarity (430 mOsm/kg for 8 h, followed by 550 mOsm/kg for 16 h). The role of ERK1/2 and p38MAPK pathways were determined by their inhibitors U0126 and SB202190 respectively. The expression of SOX9 and downstream aggrecan and collagen II, biochemical content, and histology were used to assess NP matrix synthesis. The findings revealed that NP matrix synthesis was promoted in iso‐ and cyclic‐osmolarity cultures compared to hypo‐ or hyper‐osmolarity culture although the level of matrix synthesis in cyclic‐osmolarity culture did not reach that in iso‐osmolarity culture. Further analysis suggested that inhibition of the ERK1/2 or p38MAPK pathway in iso‐ and cyclic‐osmolarity cultures reduced NP matrix production. Therefore, we concluded that the effects of osmolarity on NP matrix synthesis depend on osmolarity level (hypo‐, iso‐, or hyper‐) and osmolarity mode (constant or cyclic), and the ERK1/2 and p38MAPK pathways may participate in this process. © 2015 Orthopaedic Research Society. Published by Wiley Periodicals, Inc. J Orthop Res 34:1092–1100, 2016.     

Alterations in intervertebral disc composition,matrix homeostasis and biomechanical behavior in the UCD‐T2DM rat model of type 2 diabetes

Type 2 diabetes (T2D) adversely affects many tissues, and the greater incidence of discogenic low back pain among diabetic patients suggests that the intervertebral disc is affected too. Using a rat model of polygenic obese T2D, we demonstrate that diabetes compromises several aspects of disc composition, matrix homeostasis, and biomechanical behavior. Coccygeal motion segments were harvested from 6‐month‐old lean Sprague‐Dawley rats, obese Sprague‐Dawley rats, and diabetic obese UCD‐T2DM rats (diabetic for 69 ± 7 days). Findings indicated that diabetes but not obesity reduced disc glycosaminoglycan and water contents, and these degenerative changes correlated with increased vertebral endplate thickness and decreased endplate porosity, and with higher levels of the advanced glycation end‐product (AGE) pentosidine. Consistent with their diminished glycosaminoglycan and water contents and their higher AGE levels, discs from diabetic rats were stiffer and exhibited less creep when compressed. At the matrix level, elevated expression of hypoxia‐inducible genes and catabolic markers in the discs from diabetic rats coincided with increased oxidative stress and greater interactions between AGEs and one of their receptors (RAGE). Taken together, these findings indicate that endplate sclerosis, increased oxidative stress, and AGE/RAGE‐mediated interactions could be important factors for explaining the greater incidence of disc pathology in T2D. © 2015 Orthopaedic Research Society. Published by Wiley Periodicals, Inc. J Orthop Res 33:738–746, 2015.     

Development of a bovine decellularized extracellular matrix‐biomaterial for nucleus pulposus regeneration

Painful intervertebral disc (IVD) degeneration is a common cause for spinal surgery. There is a clinical need to develop injectable biomaterials capable of promoting IVD regeneration, yet many available biomaterials do not mimic the native extracellular matrix (ECM) or promote matrix production. This study aimed to develop a decellularized injectable bovine ECM material that maintains structural and compositional features of native tissue and promotes nucleus pulposus (NP) cell (NPC) and mesenchymal stem cell (MSC) adaption. Injectable decellularized ECM constructs were created using 3 NP tissue decellularization methods (con.A: sodium deoxycholate, con.B: sodium deoxycholate & sodium dodecyl sulfate, con.C: sodium deoxycholate, sodium dodecyl sulfate & TritonX‐100) and evaluated for protein, microstructure, and for cell adaptation in 21 day human NPC and MSC culture experiments. Con.A was most efficient at DNA depletion, preserved best collagen microstructure and content, and maintained the highest glycosaminoglycan (GAG) content. NPCs in decellularized constructs of con.A&B demonstrated newly synthesized GAG production, which was apparent from “halos” of GAG staining surrounding seeded NPCs. Con.A also promoted MSC adaption with high cell viability and ECM production. The injectable decellularized NP biomaterial that used sodium deoxycholate without additional decellularization steps maintained native NP tissue structure and composition closest to natural ECM and promoted cellular adaptation of NP cells and MSCs. This natural decellularized biomaterial warrants further investigation for its potential as an injectable cell seeded supplement to augment NP replacement biomaterials and deliver NPCs or MSCs. © 2015 Orthopaedic Research Society. Published by Wiley Periodicals, Inc. J Orthop Res 34:876–888, 2016.     

PDGF‐BB inhibits intervertebral disc cell apoptosis in vitro

Degeneration of the intervertebral disc (IVD) results in deterioration of the spinal motion segment and can lead to debilitating back pain. Given the established mitotic and anti‐apoptotic effects of recombinant human platelet‐derived growth factor‐BB (rhPDGF‐BB) in a variety of cell types we postulated that rhPDGF‐BB might delay disc cell degeneration through inhibition of apoptosis. To address this hypothesis, we treated human IVD cells isolated from five independent patients with rhPDGF‐BB in monolayer and 3D pellet cultures. The anti‐apoptotic potential, cell proliferative capacity, morphology/pellet differentiation, and gene expression of PDGF‐treated IVD cells were evaluated via flow cytometry/immunohistochemistry, MTT assays, histology, and quantitative RT‐PCR, respectively. We found that rhPDGF‐BB treatment significantly inhibited cell apoptosis, increased cell proliferation and matrix production, and maintained mRNA expression of critical extracellular matrix genes. This study suggests two possible mechanisms for the anti‐degenerative effects of rhPDGF‐BB on human IVD cells. First, PDGF treatment strongly inhibited IVD cell apoptosis in 3D cultures. Second, rhPDGF‐BB acts as an anabolic agent, promoting maintenance of IVD cell phenotype in 3D culture, based on the molecular and protein expression analysis. We speculate that rhPDGF‐BB may be used as a biologic treatment to target early degenerative IVD disease in the future. © 2014 Orthopaedic Research Society. Published by Wiley Periodicals, Inc. J Orthop Res 32:1181–1188, 2014.     

Low oxygen tension stimulates the redifferentiation of dedifferentiated adult human nasal chondrocytes

OBJECTIVE: To determine the effect of dissolved oxygen tension (DO) on the redifferentiation of dedifferentiated adult human nasal septum chondrocytes cultured as pellets. DESIGN: After isolation, human nasal chondrocytes were expanded in monolayer culture, which resulted in their dedifferentiation. Dedifferentiated cells were pelleted, transferred to a bioreactor and maintained for up to 21 days at 100% DO (21% oxygen), 25% DO (5.25% oxygen) or 5% DO (1% oxygen), which was controlled in the liquid phase. Redifferentiation was assessed by staining the extracellular matrix with safranin-O and by the immunolocalization of collagen types I, II, IX and of a fibroblast membrane marker (11-fibrau). In addition, glycosaminoglycans (GAG) and DNA content were determined spectrophotometrically. RESULTS: In monolayer culture, cells dedifferentiated and multiplied 90- to 100-fold. Cell pellets cultured in a bioreactor under conditions of low oxygen tension (25% DO or 5% DO) stained intensely for GAGs and for collagen type II, but very weakly for collagen type I. After 14 days of culturing, cell pellets maintained at 5% DO stained more intensely for collagen IX and more weakly for 11-fibrau than did those incubated at 25% DO. After 21 days of culturing the GAG content of cell pellets maintained at 5% DO was significantly greater than that of those incubated at 25% DO. Under air-saturated conditions (100% DO), the DNA and GAG contents of cell pellets decreased with time in culture. After 21 days of culturing, both parameters were substantially lower in cell pellets maintained at 100% DO than in those incubated at low oxygen tensions. The staining signals for collagen types II and IX were much weaker, and those for the markers of dedifferentiation (collagen type I and 11-fibrau) much stronger under air-saturated conditions than at low oxygen tensions. CONCLUSION: These observations demonstrate that using the present set-up, low oxygen tension stimulates the redifferentiation of dedifferentiated adult human nasal chondrocytes in pellet cultures.     

Modulation of bovine articular chondrocyte gene expression in vitro by oxygen tension

OBJECTIVE: Adult articular cartilage is a physiologically hypoxic tissue with a proposed gradient of oxygen tension ranging from <10% oxygen at the cartilage surface to <1% in the deepest layers. This gradient may be disturbed during diseases of the joint, for example in rheumatoid arthritis when synovial fluid pO(2)falls. We investigated whether changes in oxygen tension modulate gene expression in articular chondrocytes. DESIGN: Bovine articular chondrocytes were cultured in alginate beads in medium maintained at <0.1, 5, 10 or 20% oxygen. A modified RNA arbitrarily primed polymerase chain reaction (RAP-PCR) technique was used to identify several genes whose mRNA abundance in articular chondrocytes was dependent upon oxygen tension. Northern hybridization slot blots were used to quantify changes in mRNA level relative to a housekeeping gene, beta-actin. RESULTS: Genes found by RAP-PCR to undergo up-regulation in hypoxia included TIMP-1 and integrin-linked kinase. Collagen V mRNA levels were down-regulated in hypoxic chondrocytes. This led us to examine mRNA levels for various cytokines, matrix structural molecules and beta1 integrin. Interleukin 1beta, transforming growth factor beta and connective tissue growth factor were all up-regulated by low oxygen tensions, as was beta1 integrin. Collagen II (COL2A1) was down-regulated by hypoxia but aggrecan mRNA levels remained unchanged. The mRNA levels for GAPDH, the archetypal hypoxia responsive gene, were not modulated in articular chondrocytes by changes in oxygen tension. CONCLUSIONS: Oxygen tension modulates the abundance of mRNAs encoding structural molecules, several cytokines, beta1 integrin and integrin-linked kinase in articular chondrocytes. This may be important during disease progression. Chondrocytes are unusual in their response to hypoxia, presumably because they exist physiologically in a low oxygen environment.     

RNA sequencing identifies gene regulatory networks controlling extracellular matrix synthesis in intervertebral disk tissues

Degenerative disk disease of the spine is a major cause of back pain and disability. Optimization of regenerative medical therapies for degenerative disk disease requires a deep mechanistic understanding of the factors controlling the structural integrity of spinal tissues. In this investigation, we sought to identify candidate regulatory genes controlling extracellular matrix synthesis in spinal tissues. To achieve this goal we performed high throughput next generation RNA sequencing on 39 annulus fibrosus and 21 nucleus pulposus human tissue samples. Specimens were collected from patients undergoing surgical discectomy for the treatment of degenerative disk disease. Our studies identified associations between extracellular matrix genes, growth factors, and other important regulatory molecules. The fibrous matrix characteristic of annulus fibrosus was associated with expression of the growth factors platelet derived growth factor beta (PDGFB), vascular endothelial growth factor C (VEGFC), and fibroblast growth factor 9 (FGF9). Additionally we observed high expression of multiple signaling proteins involved in the NOTCH and WNT signaling cascades. Nucleus pulposus extracellular matrix related genes were associated with the expression of numerous diffusible growth factors largely associated with the transforming growth signaling cascade, including transforming factor alpha (TGFA), inhibin alpha (INHA), inhibin beta A (INHBA), bone morphogenetic proteins (BMP2, BMP6), and others. Clinical significance: this investigation provides important data on extracellular matrix gene regulatory networks in disk tissues. This information can be used to optimize pharmacologic, stem cell, and tissue engineering strategies for regeneration of the intervertebral disk and the treatment of back pain. © 2017 Orthopaedic Research Society. Published by Wiley Periodicals, Inc. J Orthop Res 36:1356–1369, 2018.

     

Redifferentiation of dedifferentiated bovine articular chondrocytes in alginate culture under low oxygen tension.

OBJECTIVE: To determine the influence of low oxygen tension on the redifferentiation and matrix production of dedifferentiated articular chondrocytes in monolayer and alginate bead culture. METHODS: Bovine articular chondrocytes were isolated enzymatically. After multiplication and dedifferentiation in a 2-week monolayer culture under 21% oxygen, the cells were subcultured in monolayer or alginate bead culture and subjected to 21% or 5% O(2)for 2 or 3 weeks in order to redifferentiate. Controls consisted of primary cultures in alginate. Matrix production was monitored immunocytochemically [collagen types I, II, IX, and GAGs (keratan sulfate, chondroitin-4- and -6-sulfate)] and collagen type II additionally assayed by Western blotting. Biosynthetic activity was measured by [(3)H]-proline incorporation and cell-viability by the trypan blue exclusion method. RESULTS: The cell number increased more than four-fold during dedifferentiation. Collagen type II was not produced by dedifferentiated chondrocytes under 5% or 21% oxygen in the monolayers or under 21% in alginate. However, dedifferentiated cells in alginate subjected to 5% oxygen exhibited a strong collagen type II expression indicating a redifferentiation. Additionally, collagen type IX and GAGs were also higher and [(3)H]-proline incorporation increased significantly. Primary cultures in alginate displayed a stronger collagen type II expression under 5% but no significant differences for other extracellular matrix components, or [(3)H]-proline incorporation. Viability was approximately 90% for all alginate cultures. CONCLUSION: A combination of alginate and high oxygen tension might not be suitable for redifferentiation or culturing of dedifferentiated chondrocytes. However, low oxygen tension promotes or induces a redifferentiation of dedifferentiated cells in alginate, stimulates their biosynthetic activity, and increases collagen type II production in primary alginate cultures.     

Anti‐inflammatory effect of platelet‐rich plasma on nucleus pulposus cells with response of TNF‐α and IL‐1

The purpose of this study was to investigate the anti‐inflammatory effect of platelet‐rich plasma (PRP) with collagen matrix on human nucleus pulposus (NP) cell in response to pro‐inflammatory cytokines such as tumor necrosis factor‐alpha (TNF‐α) and interleukin‐1 (IL‐1). NP cells from human disks were cultured in a monolayer and maintained in the collagen matrix prior to the addition of recombinant human IL‐1 and TNF‐α. After applying IL‐1 and TNF‐α, PRP prepared by using a commercially available platelet concentration system was added. The response was investigated using real‐time PCR for mRNA expression of type II collagen, aggrecan, matrix metalloproteinase‐3 (MMP‐3), and cyclooxygenase‐2 (COX‐2). The combination of IL‐1β and TNF‐α led to decrease of matrix synthesis gene expression such as collagen type II and aggrecan and increase of the degradation gene expression of COX‐2 and MMP‐3, compared to the control. Consecutive PRP exposure significantly recovered the down‐regulated gene expression of collagen type II and aggrecan and significantly reduced the increased MMP‐3 and COX‐2 gene expression, compared to that of control groups with pro‐inflammatory cytokines. The administration of PRP with collagen matrix markedly suppressed cytokine‐induced pro‐inflammatory degrading enzymes and mediators in the NP cell. It also rescued gene expression concerning matrix synthesis, thereby stabilizing NP cell differentiation. © 2013 Orthopaedic Research Society. Published by Wiley Periodicals, Inc. J Orthop Res 32:551–556, 2014.     

Characterization of nucleus pulposus-like tissue formed in vitro.

In order to be able to study the metabolism of nucleus pulposus (NP) tissue, we developed a cell culture system that resulted in the formation of NP-like tissue in vitro. NP cells were isolated from sheep lumbar spines and grown on filter inserts (Millicell CM). Histological examination showed that the cells accumulated extracellular matrix and formed a continuous layer of NP-like tissue. The accumulation of sulfated proteoglycans in the NP-like tissue continued up to 10 weeks and this was paralleled by an increase in tissue thickness and dry weight. DNA content remained stable during the first 4 weeks but then decreased over time. The amount of DNA, glycosaminoglycan (GAG) and collagen per mg dry weight of the tissue generated after 10 weeks in culture were 1.25+/-0.02, 301.6+/-27.7 and 411+/-65 microg, respectively, compared with 1.04+/-0.08, 320.6+/-21.2 and 399+/-4.4 microg (mean +/- SEM) for the in vivo tissue. There was no significant difference between in vitro and in vivo tissue. The cells in culture synthesized large proteoglycans (kav = 0.26+/-0.03, mean +/- S.D.) which were similar in size to those synthesized by cells in NP tissue in ex vivo culture (kav = 0.22+/-0.02, mean +/- S.D.) as determined by Sepharose CL-2B column chromatography. The in vitro generated tissue contained type II collagen as demonstrated by sodium dodecyl sulfate-polyacrylamide gel (SDS-PAGE) and silver staining as well as Western blot analysis. NP cells grown on filters generate tissue similar in composition to the in vivo tissue, for the characteristics examined to date, and should be a suitable model to use to study NP metabolism and extracellular matrix turnover.     

Wound hypoxia in deep tissue after incision in rats

Our previous studies using rat models of incisional pain have shown that tissue lactate levels increase and pH decreases for several days after incision, suggesting the presence of an ischemic‐like condition. The purpose of this study was to evaluate the time course and the extent of tissue hypoxia that develops in incised muscle and skin. We directly measured oxygen tension at several time points after incisions of the gastrocnemius muscle, the paraspinal skin, and the plantar hindpaw in anesthetized rats using an oxygen‐sensitive microelectrode. In vivo hypoxia of the incised tissues was also evaluated immunohistochemically using a hypoxia marker, pimonidazole hydrochloride. To minimize intersubject variability, unincised contralateral tissues were used as a control. Tissue oxygen tension was decreased in both skeletal muscle and skin compared with control, for several days after incision. When measured directly, oxygen tension decreased immediately and remained low for several days after incisions. Pimonidazole immunostaining revealed hypoxic areas in incised muscle and skin for several days. By postoperative day 10, tissue oxygen tension recovered to that of control tissue. These results support the evidence that a hypoxic condition is present in deep tissue after incisions and that an ischemic‐like mechanism may contribute to postoperative pain.