Intervertebral disk repair by protein, gene, or cell injection: a framework for rehabilitation-focused biologics in the spine.

Low back pain carries an enormous socioeconomic burden. Current treatment modalities for symptomatic intervertebral disk (IVD) degeneration have limited and often inconsistent clinical benefits. Novel approaches with the potential to halt or even reverse disk degeneration and restore physiologic disk function, such as biological treatments, are therefore very attractive. The following barriers are impeding the development of successful therapeutic interventions: (1) the biology and pathophysiology of disk degeneration are not well understood, and (2) the precise relationship between IVD degeneration and low back pain remains unclear. This article reviews the structural changes that take place during IVD degeneration and their relationship to diskogenic back pain. It also presents treatment modalities that currently are under laboratory investigation and are being studied in clinical trials. The authors of recent studies have shown that the content of large proteoglycans, such as aggrecan and versican, decreases with aging and IVD degeneration, whereas the content of certain small proteoglycans, such as biglycan, increases. Proinflammatory cytokines such as interleukin-1 and tumor necrosis factor-α also are associated with IVD degeneration and are potential biomarkers of IVD degeneration and repair. Our group of investigators and others have developed in vitro models of IVD cell and explant culture in addition to in vivo animal models to study IVD degeneration and repair. With the use of these models, we have tested candidate therapeutic agents to assess their therapeutic potential for matrix restoration. When a rabbit annular puncture model of IVD degeneration was used, injections of either bone morphogenetic protein-7 (also known as osteogenic protein-1) or bone morphogenetic protein-14 (also known as growth differentiation factor-5) were shown to be effective in restoring IVD structures. On the basis of these data, the Food and Drug Administration has recently allowed the initiation of Investigational New Drug clinical trials on osteogenic protein-1 and growth differentiation factor-5 in the United States. Protein therapies such as other growth factors, inhibitors of degradation enzymes or cytokines, and cell therapies also are being investigated in laboratory settings with the goal of restoring disk function and alleviating back pain symptoms. These therapies may be used by physiatrists with the skills required to administer intradiskal injections and supervise a comprehensive rehabilitation program after the procedures. Ultimately, the clinical use of any biological treatment discussed in this article would require the collective efforts of clinicians and researchers.

Primary bovine intervertebral disc cells transduced with adenovirus overexpressing 12 BMPs and Sox9 maintain appropriate phenotype.

OBJECTIVE: To confirm that primary intervertebral disc cells cultured in monolayer transduced with adenovirus maintained their phenotype, hence is an appropriate system to test gene therapy agents. DESIGN: Adult bovine nucleus pulposus and anulus fibrosus cells cultured in monolayer were transduced with adenoviruses expressing human bone morphogenetic proteins (AdBMPs) or Sox9 (AdSox9), or green fluorescence protein (AdGFP, as control). Chondrocyte phenotypic markers (e.g., type II collagen and aggrecan) and the chondrocyte hypertrophy marker (type X collagen) were measured 6 days after viral transduction by reverse-transcription polymerase chain reaction. RESULTS: Primary nucleus pulposus and anulus fibrosus cells transduced with AdBMPs, AdSox9, or adenovirus-expressing green fluorescence protein only (AdGFP, as control) continue to express healthy chondrocyte phenotypic markers and showed no evidence of the expression of the chondrocyte hypertrophy marker (type X collagen gene). Thus, we have shown that bovine nucleus pulposus and anulus fibrosus cells transduced with adenovirus overexpressing 12 different bone morphogenetic proteins or Sox9 maintain their phenotype in short-term culture. CONCLUSIONS: In this study, primary bovine intervertebral disc cells transduced with adenovirus overexpressing 12 bone morphogenetic proteins or Sox9 preserved their phenotype in short-term culture. These cells did not express the type X collagen gene, an undesirable chondrocyte hypertrophic gene that could lead to ossification. Therefore, low-passage intervertebral disc cells cultured in monolayer is an appropriate culture system to test therapeutic genes. We further suggest that these cells may also be appropriate for engineering tissues or for cell therapy for degenerative disc diseases.

Immunophenotypes of macular corneal dystrophy in India and correlation with mutations in CHST6.

PURPOSE: To determine the immunophenotypes of macular corneal dystrophy (MCD) in Indian patients and to correlate them with mutations in the carbohydrate 6-sulfotransferase (CHST6) gene. METHODS: Sixty-four patients from 53 families with MCD that were previously screened for mutations in CHST6 were included in an immunophenotype analysis. Antigenic keratan sulfate (AgKS) in serum as well as corneal tissue was evaluated in 31 families. Only cornea was evaluated in 11 families, and only serum was evaluated in 11 families. AgKS was detected in formalin-fixed, paraffin-embedded corneal sections by immunohistochemistry and in serum by ELISA using a monoclonal antibody against sulfated forms of KS in patients with MCD as well as normal controls. RESULTS: Analysis of corneal and/or serum AgKS disclosed MCD type I (27 families), MCD type IA (5 families), and MCD type II (3 families) in the cases studied. An additional 10 families were either MCD type I or MCD type IA since only serum AgKS data were available. Seven families manifested atypical immunophenotypes since the corneal AgKS expression was either of MCD type I or MCD type IA, but serum AgKS levels ranged from 19 ng/ml to 388 ng/ml. More than one immunophenotype was detected amongst siblings in two families. Each immunophenotype was associated with mutational heterogeneity in CHST6. CONCLUSIONS: MCD type I was the predominant immunophenotype in the Indian population studied followed by MCD type IA and then MCD type II. We detected further immunophenotypic heterogeneity by finding atypical patterns of AgKS reactivity in a subset of families. There were no simple correlations between immunophenotypes and specific mutations in CHST6, suggesting that factors other than CHST6 mutations may be contributing to the immunophenotypes in MCD.

Age-related changes in the extracellular matrix of nucleus pulposus and anulus fibrosus of human intervertebral disc.

STUDY DESIGN: To characterize age-related changes in the matrix of human intervertebral disc (IVD) specimens, human specimens from the third to the eighth decade of life were collected and analyzed for collagen and proteoglycan (PG) composition. OBJECTIVE: To identify age-related changes in the concentration of matrix macromolecules (collagen and PGs, including the small leucine-rich PGs biglycan, decorin, fibromodulin, and lumican) in human anulus fibrosus (AF) and nucleus pulposus (NP). SUMMARY OF BACKGROUND DATA: IVD degeneration is associated with changes in the concentration and fragmentation of matrix molecules. Deciphering age-related matrix alterations may help us to better understand the regulatory mechanisms underlying IVD degeneration. METHODS: Forty-six whole IVDs were obtained from the thoracolumbar spines (T11-L5) of humans aged between 32 and 80 years. All specimens were classified as Thompson grade 1 or 2 according to MRI criteria. Specimens were separated into (i) outer-and (ii) inner AF, and (iii) NP. DNA, collagen, and PG contents were measured using chemical assays, whereas small nonaggregating PG levels were analyzed by comparative Western blotting. RESULTS: Total PG and collagen contents in both the AF and NP consistently decreased with aging. The concentrations of small nonaggregating PGs varied. In the outer anulus, decorin levels decreased, whereas biglycan and fibromodulin levels increased with age. In the inner anulus and nucleus, biglycan demonstrated a significant increase with aging. These changes differed in most cases from those previously reported for degenerating disc tissues. CONCLUSION: Collagen and PGs appeared to undergo specific age-related changes in the human IVD. Although the total contents of these 2 families of molecules decreased during aging, individual species of small nonaggregating PGs showed species-specific age-related changes. Interestingly, the level of biglycan rose and remained elevated in all 3 compartments of the disc with aging. The functional significance of these alterations is yet to be determined.

Biological treatment for degenerative disc disease: implications for the field of physical medicine and rehabilitation.

Spine care is a fast-growing sector of the outpatient practice for physiatrists. Current nonsurgical treatment modalities and surgical options for severe symptomatic intervertebral disc degeneration have limited and inconsistent clinical results. Thus, the development of novel approaches, such as biological treatments that offer the potential to halt or even reverse disc degeneration and restore physiologic disc function, are very attractive. In this article, we first review the structural changes that occur during intervertebral disc degeneration and their relationship with discogenic back pain. Subsequently, we review the treatment approaches currently under clinical trial and laboratory investigation. Physiatrists specializing in spine care have the skill set required for administering intradiscal injections and supervising a comprehensive rehabilitation program after the procedures. Ultimately, the clinical use of any biological treatment discussed herein would require the collective efforts of physicians (such as physiatrists and surgeons) and researchers (such as chemical and biomedical engineers, biologists, and chemists).

Cell therapy using articular chondrocytes overexpressing BMP-7 or BMP-10 in a rabbit disc organ culture model.

STUDY DESIGN: Rabbit knee articular chondrocytes overexpressing human growth factors were injected into cultured intervertebral disc explants. Survival of the injected cells and accumulation of extracellular matrix were assessed. OBJECTIVE: To define the utility of cell-based gene delivery approach for repair of the intervertebral disc. SUMMARY OF BACKGROUND DATA: Back pain associated with symptomatic disc degeneration is a common clinical condition. Growth factors stimulate disc cell metabolism, but the ideal method for in vivo delivery has not been established. Cells as a vehicle for delivering growth factors to the disc offer potential advantages, including prolonged production of the growth factor within the disc and vital cells to participate in the repair process. METHODS: New Zealand white rabbit articular chondrocytes transduced with adenovirus expressing human bone morphogenetic protein-7 and green fluorescence protein (GFP) (AdhBMP-7), human bone morphogenetic protein-10 and GFP (AdBMP-10), or GFP alone (AdGFP, as a control) were injected into whole disc explants. Discs were maintained in culture for 1 to 2 months. At the conclusion of the culture periods, cell survival was assessed by fluorescence microscopy and extracellular matrix accumulation was assessed with biochemical methods. RESULTS: Chondrocytes achieved long-term survival in the cultured disc explants. The discs treated with chondrocytes/BMP-7 demonstrated a 50% increase in proteoglycan content within the nucleus pulposus compared to control (chondrocytes/GFP), while discs injected with chondrocytes/BMP-10 failed to show a significant increase in proteoglycan accumulation. CONCLUSION: Our study demonstrates the ability of transduced articular chondrocytes to survive and promote proteoglycan accumulation when transplanted into the intervertebral disc. These data support the potential of a cell-based gene therapy approach for disc repair. Further studies using this approach in animal models are indicated as a step towards achieving disc repair in humans.

Effect of osteogenic protein-1 on the matrix metabolism of bovine tendon cells.

Tendon rupture is a common sports injury in adults. However, the mechanical properties of repair tissue are inferior to those of normal tissue. To accelerate tendon healing, an in vivo approach using growth factors has been applied and has shown evidence for the efficacy of biological stimulation of the repair process. Recombinant human osteogenic protein-1 (rhOP-1) has been shown to be effective in stimulating matrix production by various connective tissues. To test the effect of rhOP-1 on the matrix metabolism of tendon cells in vitro, bovine tendon cells were cultured in monolayer with various doses of rhOP-1 for 7 days. The addition of rhOP-1 to cell culture media resulted in significant increases in cell proliferation, DNA content, and the synthesis of proteoglycans (PGs) and collagen, compared to control cultures. The relative percentage of large PGs in the OP-1 culture was higher than that in the control culture. In conclusion, we show for the first time that rhOP-1 stimulates the proliferation of tendon cells and their ability to synthesize and accumulate PGs and collagen in their extracellular matrix. These biological properties may be used in the tissue-engineering of tendon tissues.

Basic fibroblast growth factor accelerates matrix degradation via a neuro-endocrine pathway in human adult articular chondrocytes.

Pain-related neuropeptides released from synovial fibroblasts, such as substance P, have been implicated in joint destruction. Substance P-induced inflammatory processes are mediated via signaling through a G-protein-coupled receptor, that is, neurokinin-1 tachykinin receptor (NK(1)-R). We determined the pathophysiological link between substance P and its receptor in human adult articular cartilage homeostasis. We further examined if catabolic growth factors such as basic fibroblast growth factor (bFGF or FGF-2) or IL-1beta accelerate matrix degradation via a neural pathway upregulation of substance P and NK(1)-R. We show here that substance P stimulates the production of cartilage-degrading enzymes, such as matrix metalloproteinase-13 (MMP-13), and suppresses proteoglycan deposition in human adult articular chondrocytes via NK(1)-R. Furthermore, we have demonstrated that substance P negates proteoglycan stimulation promoted by bone morphogenetic protein-7, suggesting the dual role of substance P as both a pro-catabolic and anti-anabolic mediator of cartilage homeostasis. We report that bFGF-mediated stimulation of substance P and its receptor NK(1)-R is, in part, through an IL-1beta-dependent pathway.

Regulation of immature cartilage growth by IGF-I, TGF-beta1, BMP-7, and PDGF-AB: role of metabolic balance between fixed charge and collagen network.

Cartilage growth may involve alterations in the balance between the swelling tendency of proteoglycans and the restraining function of the collagen network. Growth factors, including IGF-I, TGF-beta1, BMP-7, and PDGF-AB, regulate chondrocyte metabolism and, consequently, may regulate cartilage growth. Immature bovine articular cartilage explants from the superficial and middle zones were incubated for 13 days in basal medium or medium supplemented with serum, IGF-I, TGF-beta1, BMP-7, or PDGF-AB. Variations in tissue size, accumulation of proteoglycan and collagen, and tensile properties were assessed. The inclusion of serum, IGF-I, or BMP-7 resulted in expansive tissue growth, stimulation of proteoglycan deposition but not of collagen, and a diminution of tensile integrity. The regulation of cartilage metabolism by TGF-beta1 resulted in tissue homeostasis, with maintenance of size, composition, and function. Incubation in basal medium or with PDGF-AB resulted in small volumetric and compositional changes, but a marked decrease in tensile integrity. These results demonstrate that the phenotype of cartilage growth, and the associated balance between proteoglycan content and integrity of the collagen network, is regulated differentially by certain growth factors.

Recombinant human osteogenic protein-1 upregulates proteoglycan metabolism of human anulus fibrosus and nucleus pulposus cells.

STUDY DESIGN: In vitro assessment of the effects of recombinant human osteogenic protein-1 (rhOP-1) on the proteoglycan metabolism of human intervertebral disc cells. OBJECTIVES: To determine whether rhOP-1 is effective in stimulating the cell proliferation and proteoglycan metabolism of human intervertebral disc cells cultured in alginate beads. SUMMARY OF THE BACKGROUND DATA: OP-1 has been shown to stimulate the proteoglycan and collagen synthesis of rabbit intervertebral disc cells in vitro. In vivo, a single injection of rhOP-1 restored the disc height of a degenerated disc in the rabbit anular-puncture model. The effect of rhOP-1 on human intervertebral disc cells remains unknown. METHODS: Human nucleus pulposus and anulus fibrosus cells were isolated from the discs of 4 cadaveric spines and one surgical specimen. After preculture for 7 days, alginate beads containing nucleus pulposus and anulus fibrosus cells were cultured for 21 days in media containing 10% fetal bovine serum with 0, 100, or 200 ng/mL rhOP-1 and supplements. The synthesis and accumulation of proteoglycans and the DNA content were biochemically assessed. RESULTS: The addition of rhOP-1 to the media resulted in the prevention of a decreased cell number during culture. Treatment with rhOP-1, compared with the control condition (10% fetal bovine serum), significantly upregulated proteoglycan synthesis and accumulation in alginate beads in all cases tested. A longer exposure over 14 days to rhOP-1 resulted in a pronounced response. The retention of newly-synthesized proteoglycan was higher in the rhOP-1-treated cells than in the control. CONCLUSIONS: rhOP-1 was effective in stimulating the cell proliferation and proteoglycan metabolism of human intervertebral disc cells in vitro. The results supported the hypothesis that an in vivo injection of rhOP-1 may increase the metabolic activity of disc cells or prevent apoptosis of disc cells in a degenerated disc. However, the requirement for a long exposure to rhOP-1 for human cells may suggest the need for a prolonged supply of rhOP-1 by a drug delivery system or by repeated injections.

Mechanisms of cartilage growth: modulation of balance between proteoglycan and collagen in vitro using chondroitinase ABC.

OBJECTIVE: To examine the cartilage growth-associated effects of a disruption in the balance between the swelling pressure of glycosaminoglycans (GAGs) and the restraining function of the collagen network, by diminishing GAG content prior to culture using enzymatic treatment with chondroitinase ABC. METHODS: Immature bovine articular cartilage explants from the superficial and middle layers were analyzed immediately or after incubation in serum-supplemented medium for 13 days. Other explants were treated with chondroitinase ABC to deplete tissue GAG and also either analyzed immediately or after incubation in serum-supplemented medium for 13 days. Treatment- and incubation-associated variations in tissue volume, contents of proteoglycan and collagen network components, and tensile mechanical properties were assessed. RESULTS: Incubation in serum-supplemented medium resulted in expansive growth with a marked increase in tissue volume that was associated with a diminution of tensile integrity. In contrast, chondroitinase ABC treatment on day 0 led to a marked reduction of GAG content and enhancement of tensile integrity, and subsequent incubation led to maturational growth with minimal changes in tissue volume and maintenance of tensile integrity at the enhanced levels. CONCLUSION: The data demonstrate that a manipulation of GAG content in articular cartilage explants can distinctly alter the growth phenotype of cartilage. This may have practical utility for tissue engineering and cartilage repair. For example, the expansive growth phenotype may be useful to fill cartilage defects, while the maturational growth phenotype may be useful to induce matrix stabilization after filling defect spaces.

Comparative effects of bone morphogenetic proteins and sox9 overexpression on extracellular matrix metabolism of bovine nucleus pulposus cells.

STUDY DESIGN: An in vitro biologic study of the effects of adenovirus expressing bone morphogenetic proteins (BMPs) and adenovirus expressing Sox9 on extracellular matrix metabolism by bovine nucleus pulposus cells. OBJECTIVE: To compare the effects of recombinant adenoviral vectors expressing various BMPs (2, 3, 4, 5, 7, 8, 10, 11, 12, 13, 14, and 15) and Sox9 on extracellular matrix accumulation by bovine nucleus pulposus cells. SUMMARY OF BACKGROUND DATA: Nucleus pulposus matrix production may be promoted by transducing the cells with genes that permit the sustained expression of growth factors. The choice of the particular factors or BMPs to be studied for these applications has been largely based on the commercial availability of such products. To our knowledge, this study is the first effort to evaluate systematically the relative effectiveness of the various members of the BMP family in promoting intervertebral disc matrix repair. METHODS: Adult bovine nucleus pulposus cells cultured in monolayer were transduced with adenoviruses expressing human BMP-2, 3, 4, 5, 7, 8, 10, 11, 12, 13, 14, and 15, and adenovirus expressing Sox9. Proteoglycan and collagen accumulation, and cell proliferation were measured 6 days after viral transduction. As a positive control, cells were cultured without any exogenous gene in the presence of recombinant human (rh)BMP-7. RESULTS: Nucleus pulposus cells transduced with adenoviruses expressing BMP-2, 3, 4, 5, 7, 8, 10, 13, 15, and Sox9 accumulated more proteoglycans than nucleus pulposus cells transduced with adenovirus expressing green fluorescent protein (control). It is noteworthy that nucleus pulposus cells transduced with adenoviruses expressing BMP-2 and 7 resulted in essentially as great a stimulation of proteoglycan accumulation as nucleus pulposus cells maintained in the presence of rhBMP-7 (adenoviruses expressing BMP-2: 104% increase; adenoviruses expressing BMP-7: 162% increase; and rhBMP-7: 120% increase). Nucleus pulposus cells transduced with BMP-2, 4, 5, 7, 8, 10, 14, 15, and Sox9 accumulated significantly more collagen compared to nucleus pulposus cells transduced with adenovirus expressing green fluorescent protein; adenoviruses expressing BMP-4 and 14 were the most effective (552% and 661% increase, respectively). Nucleus pulposus cells also proliferated, as measured by deoxyribonucleic acid content, when transduced with adenoviruses expressing BMP-2 and 8. CONCLUSIONS: To our knowledge, for the first time, we have shown the relative effectiveness of 12 different BMPs and Sox9 in stimulating proteoglycan and collagen production by nucleus pulposus cells. Adenoviruses expressing BMP-2 and 7 were the most effective in stimulating proteoglycan accumulation, while adenoviruses expressing BMP-4 and 14 were the most effective in stimulating collagen accumulation. To our knowledge, this study is the first to compare the relative effectiveness of various BMPs and Sox9 on extracellular matrix accumulation by nucleus pulposus cells, and could help to develop more efficacious approaches to the treatment of degenerating intervertebral discs.

Osteogenic protein 1 in synovial fluid from patients with rheumatoid arthritis or osteoarthritis: relationship with disease and levels of hyaluronan and antigenic keratan sulfate.

The measurement of body fluid levels of biochemical markers in joint tissues has begun to provide clinically useful information. Synovial fluid (SF) plays an important role in articular joint lubrication, nutrition, and metabolism of cartilage and other connective tissues within the joint. The purpose of our study was to identify and characterize osteogenic protein 1 (OP-1) in SF from patients with rheumatoid arthritis (RA) or with osteoarthritis (OA) and to correlate levels of OP-1 with those of hyaluronan (HA) and antigenic keratan sulfate (AgKS). SF was aspirated from the knees of patients with either RA or OA and from the knees of asymptomatic organ donors with no documented history of joint disease. The presence of detectable OP-1 in SF was demonstrated by western blots with specific anti-pro-OP-1 and anti-mature OP-1 antibodies. Measurement of levels of OP-1, HA and AgKS was performed using ELISAs. OP-1 was identified in human SF in two forms, pro-OP-1 and active (mature) OP-1--mature OP-1 being detected only in SF from OA patients and RA patients. Levels of OP-1 and HA were higher in RA patients than in OA patients and asymptomatic donors, while the level of AgKS was highest in SF from asymptomatic donors. Statistically significant differences were found between SF levels of OP-1 in RA and OA patients and between SF levels of AgKS among the three groups tested. The SF content of OP-1 tended to correlate positively with HA levels, but negatively with AgKS concentrations. In conclusion, the results of this study suggest that measurement of OP-1 in joint fluid may have value in the clinical evaluation of joint disease processes.

Tracking chondrocytes and assessing their proliferation with PKH26: effects on secretion of proteoglycan 4 (PRG4).

Distinguishing between implanted and host-derived cells, as well as between distinct cell phenotypes, would be useful in assessing the mechanisms of cell-based repair of cartilage. The fluorescent tracker dye, PKH26, was previously applied to several cell types to assess proliferation in vitro and to track cells in vivo. The objectives of this study were to assess the utility of PKH26 for tracking chondrocytes from superficial and middle zones and their proliferation, and determine the effects of PKH26 on chondrocyte functions, in particular, proliferation and secretion of Proteoglycan 4 (PRG4). PKH26-labeled and unlabeled superficial and middle zone chondrocytes were plated in either low- or high-density monolayer culture and analyzed for retention of PKH26 by flow cytometry and fluorescence microscopy at days 0 and 7. Cell suspensions and conditioned media were analyzed for DNA and secretion of PRG4, respectively. Flow cytometric histograms were deconvolved so that the number of cells in each doubling generation contributing to the final cell population could be estimated. Chondrocytes were consistently and intensely labeled with PKH26 through 7 cycles of division. At day 7 of culture, >97% of superficial zone cells seeded at low or high density could be distinguished as fluorescent, as could middle zone cells seeded at high density. Retention of cell fluorescence after PKH26 labeling and lack of adverse effects on cell proliferation and synthesis of PRG4 suggest that PKH26 can be useful in determining the fate and function of implanted chondrocytes in vivo, as well as monitoring proliferation in vitro.

Platelet-rich plasma (PRP) stimulates the extracellular matrix metabolism of porcine nucleus pulposus and anulus fibrosus cells cultured in alginate beads.

STUDY DESIGN: In vitro assessment of the effects of platelet-rich plasma on the extracellular matrix metabolism of porcine intervertebral disc cells. OBJECTIVES: To determine whether platelet-rich plasma is effective in stimulating cell proliferation and extracellular matrix metabolism by porcine disc cells cultured in alginate beads. SUMMARY OF BACKGROUND DATA: Platelet-rich plasma is used to accelerate wound healing and tissue regeneration. Activated platelets release multiple growth factors that regulate cell proliferation, differentiation, and morphogenesis. Individual growth factors present in platelet-rich plasma have been demonstrated to affect the metabolism of intervertebral disc cells. METHODS: Platelet-poor and platelet-rich plasma was isolated from fresh porcine blood using a commercially available platelet concentration system. After preculture for 7 days and serum starvation for 24 hours, the beads containing nucleus pulposus and anulus fibrosus cells were then cultured for another 72 hours in serum-free medium, 10% fetal bovine serum, 10% platelet-poor plasma, or 10% platelet-rich plasma. The synthesis of proteoglycans and collagen, the accumulation of proteoglycans, and the DNA content were biochemically assessed. RESULTS: Platelet-rich plasma had a mild stimulatory effect on cell proliferation of intervertebral disc cells. Platelet-rich plasma treatment significantly upregulated proteoglycan and collagen synthesis and proteoglycan accumulation when compared with platelet-poor plasma. CONCLUSIONS: Platelet-rich plasma was effective in stimulating cell proliferation and extracellular matrix metabolism. The response to platelet-rich plasma was greater in the case of anulus fibrosus cells than of nucleuspulposus cells. The local administration of platelet-rich plasma might stimulate intervertebral disc repair. In addition, given the risks of using animal serum for tissue engineering, autologous blood may gain favor as a source of growth factors and serum supplements needed for stimulating cells to engineer intervertebral disc tissues.

Serum and synovial fluid concentrations of keratan sulfate and hyaluronan in dogs with induced stifle joint osteoarthritis following cranial cruciate ligament transection.

OBJECTIVE: To examine longitudinal changes in serum and synovial fluid concentrations of keratan sulfate (KS) and hyaluronan (HA) after cranial cruciate ligament (CCL) transection in dogs. ANIMALS: 12 clinically normal adult mixed-breed dogs. PROCEDURE: Following CCL transection in the right stifle joint, KS and HA concentrations were determined in serum and neat (undiluted) synovial fluid prior to and 1, 2, 3, and 12 months after surgery. Postsurgical dilution of synovial fluid was corrected by use of urea as a passive marker. RESULTS: Synovial fluid KS and HA concentrations decreased at 1, 2, and 3 months after surgery in operated stifle joints, compared with baseline values. Synovial fluid KS concentration decreased in unoperated stifle joints at 1 month. A decrease in synovial fluid KS concentration was found in operated stifle joints, compared with unoperated stifle joints, at 2 and 3 months, and a decrease in synovial fluid HA concentrations was also found in operated stifle joints, compared with unoperated stifle joints, at 1, 2, and 3 months. Serum KS concentrations increased from baseline values at 3 months after surgery. Hyaluronan concentrations in operated stifle joints were lower than baseline values at 1, 2, and 3 months. Urea-adjusted synovial fluid concentrations revealed that dilution did not account for the decline in biomarker concentrations. CONCLUSIONS AND CLINICAL RELEVANCE: The initial decrease and subsequent increase in synovial fluid concentrations of HA and KS may be caused by an acute inflammatory response to surgical intervention that negatively affects cartilage metabolism or an increase in production of immature proteoglycans.

Transduced bovine articular chondrocytes affect the metabolism of cocultured nucleus pulposus cells in vitro: implications for chondrocyte transplantation into the intervertebral disc.

STUDY DESIGN: Biologic study on the effects of coculture of bovine articular chondrocytes transduced ex vivo with genes expressing bone morphogenetic proteins (BMPs) on nucleus pulposus (NP) cells. OBJECTIVE: To evaluate the effects of bovine articular chondrocytes transduced with adenoviruses expressing various BMPs on proteoglycan and collagen production, and cellular proliferation of NP cells in vitro. SUMMARY OF BACKGROUND DATA: Matrix synthesis by intervertebral disc cells is promoted by exposing the cells to growth factors or delivering genes that permit sustained expression of growth factors. We propose a novel therapeutic approach involving delivery of autologous chondrocytes, transduced ex vivo with bioactive proteins, to provide both the cells and proteins required to stimulate disc healing. METHODS: Adult bovine articular chondrocytes were transduced with adenoviruses (Ads) expressing either BMP-2, 4, 5, 7, 10, or 13 and plated as monolayers. Bovine NP cells encapsulated in alginate beads were cocultured, floating in the medium. Proteoglycan and collagen accumulation, and NP cell proliferation were measured after 6 days of coculture. As a positive control, beads were cocultured with articular chondrocytes in the presence of rhBMP-7. RESULTS: NP cells cocultered with articular chondrocytes transduced with BMPs-2, 4, 7, and 10 accumulated significantly (P < 0.05) more proteoglycan than when cocultured with chondrocytes transduced with AdGFP (control) [AdBMP-2: 23.6%; AdBMP-4: 27.0%; AdBMP-7: 129.1%; AdBMP-10: 102.1% increases respectively]. Collagen accumulation was significantly (P < 0.05) increased by NP cells cocultured with articular chondrocytes transduced with BMPs-2, 4, 5, and 7. [AdBMP-2: 104.6%; AdBMP-4: 40.6%; AdBMP-5: 58.6%; AdBMP-7: 55.5% increases respectively]. NP cells proliferated when cocultured with articular chondrocytes transduced with AdBMP-2 and -7. CONCLUSIONS: Bovine NP cells are stimulated to produce proteoglycans and collagen when exposed to chondrocytes transduced with genes for various BMPs. If applied to the treatment of disc degeneration, this strategy could provide the disc with not only metabolically active chondrocytes but also promote matrix replenishment by stimulating native NP cells.

Exposure to pulsed low intensity ultrasound stimulates extracellular matrix metabolism of bovine intervertebral disc cells cultured in alginate beads.

STUDY DESIGN: In vitro study on the effects of pulsed low intensity ultrasound on the cellular metabolism of bovine intervertebral disc cells. OBJECTIVE: To determine whether pulsed low intensity ultrasound has effects on cell proliferation and extracellular matrix metabolism by bovine intervertebral disc cells. SUMMARY OF BACKGROUND DATA: The application of pulsed low intensity ultrasound is known to be effective in stimulating fracture and cartilage repair. However, the effects of pulsed low intensity ultrasound on intervertebral disc cells are not known. METHODS: Cells of the nucleus pulposus and inner and outer anulus fibrosus were enzymatically isolated from bovine coccygeal tissue and precultured in alginate beads for 14 days. In the ultrasound group, pulsed low intensity ultrasound was administered to the culture for 20 minutes daily for an additional 20 days. The control group was cultured in the same way but without administration of ultrasound. Cell viability, DNA content, proteoglycan and collagen synthesis, and proteoglycan content at days 10 and 20 after the initiation of treatment were evaluated. Characterization of newly synthesized collagen and proteoglycan was performed. RESULTS: No significant differences in cell viability and DNA content were observed between the two groups. On day 20, proteoglycan synthesis was increased by the application of pulsed low intensity ultrasound in nucleus pulposus and inner and outer anulus fibrosus cells (24%-26% increase, P < 0.001). The application of pulsed low intensity ultrasound increased proteoglycan content in alginate beads containing inner and outer anulus fibrosus cells (P < 0.05). Collagen synthesis by cells isolated from all three zones of the intervertebral disc was increased by the application of pulsed low intensity ultrasound (16%-19% increase, P < 0.05-0.0001). CONCLUSIONS: The application of pulsed low intensity ultrasound stimulated extracellular matrix metabolism in intervertebral disc cells. Pulsed low intensity ultrasound may prove useful for the physical stimulation of cell metabolism for tissue engineering of intervertebral disc tissue.