Vyl Leung

Mesenchymal stem cell-based repair of articular cartilage with polyglycolic acid-hydroxyapatite biphasic scaffold.

This study investigates the capacity of a composite scaffold composed of polyglycolic acid-hydroxyapatite (PGA-HA) and autologous mesenchymal stem cells (MSCs) to promote repair of osteochondral defects. MSCs from culture-expanded rabbits were seeded onto a PGA and HA scaffold. After a 72-hour co-culture period, the cell-adhered PGA and HA were joined together, forming an MSCs-PGA-HA composite. Full-thickness cartilage defects in the intercondylar fossa of the femur were then implanted with the MSC-PGA-HA composite, the PGA-HA scaffold only, or they were left empty (n=20). Animals were sacrificed 16 or 32 weeks after surgery and the gross appearance of the defects was evaluated. The specimens were examined histologically for morphologic features, and stained immunohistochemically for type 2 collagen. Specimens of the MSCs-PGA-HA composite implantation group demonstrated hyaline cartilage and a complete subchondral bone formation. At 16 weeks post-implantation, significant integration of the newly formed tissue with surrounding normal cartilage and subchondral bone was observed when compared to the two control groups. At 32 weeks, no sign of progressive degeneration of the newly formed tissue was found. A significant difference in histological grading score was found compared with the control groups. The novel MSCs-seeded, PGA-HA biphasic graft facilitated both articular cartilage and subchondral bone regeneration in an animal model and might serve as a new approach for clinical applications.

Age-related degeneration of lumbar intervertebral discs in rabbits revealed by deuterium oxide-assisted MRI

OBJECTIVES Intervertebral disc (IVD) degeneration is associated with a loss of disc water content and change in biochemical composition of the disc. Rabbit is a frequently used model to evaluate the efficacy of therapeutics for disc degeneration. This study addresses whether rabbits undergo age-related disc degeneration, assessed using deuterium oxide-assisted magnetic resonance imaging (MRI) of the lumbar IVDs. MATERIALS AND METHODS The lumbar spines of adolescent, adult, and aged rabbits (6-36 months) were subjected to T2-weighted/short-tau inversion recovery (STIR) MRI scan along with water-deuterium oxide (H(2)O:D(2)O) dilutions. The total and maximum H(2)O:D(2)O index (HDi) of the lumbar IVDs were determined and compared between disc levels at different ages. RESULTS Adolescent rabbit lumbar discs had similar total HDi, suggesting the hydration and biochemical composition was similar among the lumbar levels. With the use of H(2)O:D(2)O reference, the discs were shown to undergo continual decrease in signal with aging which non-calibrated measurement method could not reveal. The HDi decrease rate was higher at the caudal than cranial levels. CONCLUSION This study provided in vivo evidence of age-related progressive disc degenerative change in rabbit lumbar discs, suggesting aged rabbits can be considered as a natural disc degeneration model in disc regeneration studies. However, it is important to select proper disc levels as intra-subject controls due to different rates of degenerative changes between caudal and cranial levels.

Mesenchymal Stem Cells Mediate Disk Regeneration by Suppression of Fibrosis in Nucleus Pulposus

Introduction Confluent fibrosis, manifested as over-accumulation of fibrous matrix during tissue repair or remodeling, leads to scar formation and often organ failure. Studies suggest that 68% of protruded intervertebral disks and 44% of prolapsed IVD exhibit signs of fibrosis in the nucleus pulposus (NP) and that disk scarring indicates advanced stages of degeneration. It is not clear how fibrosis contributes to the initiation or progression of disk degeneration, and conversely if the prevention of fibrotic events plays a role in IVD repair or regeneration. Mesenchymal stem cells (MSCs) may arrest IVD degeneration and cross-talk with IVD cells via cell-cell contact or long-range signaling, implying MSCs may modulate disk microenvironment and function indirectly. Lupine is a reliable model for IVD degeneration studies as their lumbar spine bears biological relevance to humans regarding mechanics and age-related changes. Studies showed that puncturing to the annulus could transform the NP into a fibrocartilaginous phenotype, recapitulating the fibrotic features of human disk degeneration. We here tested if MSCs can rectify IVD degeneration and its function through modulation of fibrotic events, and investigated the pathways of the action. Materials and Methods Total 6-month-old NZW rabbits with puncture-induced lumbar disk degeneration were intradiscally implanted with Chinchilla bone marrow MSCs, labeled by Qtracker and encapsulated in oligopeptide-hydrogel at 5 x 10E3 cells per disk (n = 8). Saline or hydrogel alone was injected as control. Treated disk levels were monitored by 3T MRI and radiographs up to 1 year. At 4 to 12 weeks posttreatment, the mechanical integrity of NP and joint segment were investigated by confined compression and motion stiffness tests. The collagen fibril diameter (n = 120) and the porosity of the fibril meshwork in the NP were analyzed in situ using scanning electron microscopy and morphometric analyzer. The elastic modulus of collagen fibrils (n = 75) in the NP was measured by nanoindentation using atomic force microscopy. Collagen I and II content was assessed by immunofluorescence. Human bone marrow MSC-conditioned media (MSC-CM) was collected and used to culture alginate-encapsulated NP cells harvested from patients with Pfirrmann grade III/IV disk degeneration. After 7 days, expression of fibrotic genes was studied using real-time PCR (n = 3). Results T2-weighted MRI and disk height analysis validated that MSCs arrested the degeneration. Motion segment stiffness was significantly attenuated in MSC-treated disks, indicating MSCs alleviated ankylosis caused by the degeneration and restored the spinal segment function. MSCs regenerated the swelling pressure and compressive strength of the NP, implying that MSCs regenerated joint function through preserving the biphasic tissue properties. Collagen molecules in the degenerated NP aggregated into gigantic fibrils, about 2-fold larger in diameter, resulting in fewer fibers and larger interfibrillar space. However, MSCs inhibited the deregulated higher-order assembly of fibrils. MSCs significantly reduced collagen I deposition and prevented the increase of fibril elastic modulus in the NP associated with degeneration. These suggest that the abnormal collagen fibrils are not solely resulted from fibril fusion but also a heterotypic change of collagen composition, and that the positive effects of MSCs are coupled to a regulation of fibrillogenesis in the NP matrix. MSC-CM repressed the expression of fibrosis-related genes MMP12 and HSP47 in degenerative human NP cells. Conclusion MSC transplantation can alleviate fibrotic diseases including pulmonary, renal, and cardiac fibrosis through pathways independent of MSC differentiation. Our findings imply that MSCs also rescue disk degeneration and recover disk function by suppressing fibrotic events. The action of MSCs is elicited through a regulation of fibrillogenesis in part via paracrine signaling to the degenerative NP cells. We propose a model in which MSCs reinforce the mechanical integrity of NP by normalizing the interplay between collagen meshwork and proteoglycan. Disk repair by augmenting the antifibrotic function of MSCs or use of other antifibrotic agents warrants further investigation. This study is supported by the Research Grant Council of Hong Kong and HKU Foundation. I confirm having declared any potential conflict of interest for all authors listed on this abstract Yes Disclosure of Interest None declared

Cadherin 2 as Human Notochordal Cell Marker and its Expression Pattern in Disk Degeneration

Introduction Cadherins are transmembrane glycoproteins that mediate calcium-dependent cell adhesion. Recent studies suggest that cadherins also have nonadhesive functions. Maturation of human intervertebral disk (IVD) as well as early events of IVD degeneration in a mouse model1 is associated with segregation of nucleus pulposus (NP) cells suggesting that disk degeneration may be associated with cell adhesion molecule activities. From our microarray data analysis and subsequent immunohistochemical assays, we have revealed specific expression of Cdh2 gene and its product, cadherin 2/N-cadherin, in rodent notochordal NP cells, suggesting a potential regulatory role of cadherins in IVD homeostasis. Cadherin 2 belongs to the classic cadherin family. To date, the expression and function of cadherin 2 in IVD and its relationship to disk degeneration remain elusive. We aim to study the expression pattern of cadherin 2 in human IVD using aborted fetus and clinical samples, and to investigate its function in the rodent NP via a protein ablation strategy. Materials and Methods Degenerated lumbar disks (grade 3, Schneidermans classification) (n = 5) were obtained from 45 to 58-year-old patients. Scoliotic lumbar disks (n = 5) from patients were obtained as nondegenerated samples. Lower lumbar spine of the aborted fetus in the second trimester were used as controls. Compartmental origin and degeneration status of the samples was validated by our established multicolor FAST staining method,2 in which the disk sections were sequentially stained with alcian blue, safranin-O, tartrazine, and fast green. Immunohistochemistry for cadherin 2 was performed on paraffin sections with antigen retrieval by hyaluronidase and citrate buffer method and counterstained by hematoxylin to visualize cell nuclei. The percentage of positive cells was calculated and analyzed by nonparametric u test. For protein ablation study, 4-month-old inbred Lewis rats were anesthetized and the tail IVDs were exposed for injection of rabbit anti-cadherin 2 antibody or control IgG (n = 6 for each) into the NP via 34G hypodermic needle. By 2 weeks and 8 weeks after operation, the disks were harvested for histological examination. Results Human fetal NP cells showed homogeneous cadherin 2 expression. Annulus fibrosus (AF), cartilaginous endplates, and vertebral bodies showed no signal of cadherin 2. However, adult NP showed heterogeneous cadherin 2 expression, where only a subpopulation of the NP cells expressed the protein. Nondegenerated and degenerated NP exhibited different cell distribution with the later tended to contain cell clusters. The frequency of cadherin 2 expressing cells was significantly higher in the degenerated NP (51.9%) than in nondegenerated NP (20.0%). FAST staining assay showed that degenerated NP samples were intensely stained by safranin-O instead of alcian blue, validating a loss of sulfated acidic glycosaminoglycan due to degeneration. Cadherin 2 protein ablation study showed that the vacuolated notochordal cells in the NP partially transformed into elongated or rounded nonvacuolated cells, which are commonly presented in degenerative disks. Conclusion Human fetal NP mainly contains notochordal cells. In this study, we showed that cadherin 2 is expressed homogeneously in fetal NP suggesting cadherin 2 is a marker of notochordal cells. The reduction of cadherin 2 positive NP cells in mature human IVD is consistent with gradual loss of notochordal cells after the first decade of life. Under this notion, our findings imply that a subpopulation (20%) of NP cells retain a phenotype resembling notochordal cells in mature human IVD, although morphologically they are not vacuolated. The higher frequency of cadherin 2 expressing cells in degenerated NP implicates that proliferation of cells of notochordal origin occurs during disk degeneration. Protein ablation study suggests that cadherin 2 has a regulatory function in maintenance of notochordal cells and a loss of its function may initiate disk degeneration. Overall, our study implies a function of cadherin 2 in NP homeostasis. I confirm having declared any potential conflict of interest for all authors listed on this abstract No Disclosure of Interest None declared Yang F, et al. Injury-induced sequential transformation of notochordal nucleus pulposus to chondrogenic and fibrocartilaginous phenotype in the mouse. Journal of Pathology 2009;218(1):113–121 Leung VY, et al. Matrix remodeling during intervertebral disk growth and degeneration detected by multichromatic FAST staining. Journal of Histochemistry and Cytochemistry 2009;57(3):249–256

The potential of umbilical cord derived mesenchymal stem cells in intervertebral disc repair

Introduction Nucleus pulposus (NP) is the center and major compartment of intervertebral disc (IVD). Mesenchymal stem cells (MSCs) are a type of stem cell source under intensive investigation for their potential to regenerate NP. MSCs have been identified from various sources with different characteristics. There are indications that fetal or close to fetal tissue sources contain cells with relatively undifferentiated phenotype with respect to MSCs from adult sources. Moreover, evidences have shown that umbilical cord-derived MSCs (CMSCs) may have better chondrogenic differentiation potential than bone marrow-derived MSCs (BMSCs).1 We hypothesize CMSCs might be a suitable stem cell source for NP regeneration. The aim of this research is to analyze the paracrine effect of MSCs on NP cells, and compare the effect of BMSCs and CMSCs in an attempt to identify a better MSC source for future clinical application. Materials and Methods Human BMSCs, CMSCs, and degenerated NP cells (three batches each) were isolated and characterized from patients undergoing spinal fusion and patients at caesarean delivery, respectively, after IRB approval was acquired. Conditioned media (CM) was collected after 48 hours exposure to MSC monolayer. Cell proliferation and cytotoxicity were assessed by MTT assay after 1, 3, and 7 days in MSC-CM. Proteoglycan content of NP cells in both types of MSC-CM were measured by DMMB assay after 14 days in culture. Gene expression of degeneration-related molecules of NP cells in MSC-CM, including CDH2, CD55, FBLN1, Sox9, KRT19, KRT18, and MGP, were determined by real-time RT-PCR. Protein expression of KRT19 in degenerated NP cells before and after MSC-CM treatment was examined by immunocytochemistry and confocal microscopy. All results were normalized to the control group in which the NP cells were cultured in basal medium. Results Human BMSCs and CMSCs that we isolated satisfied the minimum criteria of MSCs; that is, they were CD73(+), CD105(+), CD146(+), CD14(−), CD45(−), C34(−), and had tri-lineage differentiation potency. The overall metabolic activities of NP cells measured by MTT reading were significantly enhanced in MSC-CM than that in control basal medium, especially in CMSC-CM. This is accompanied by a slight increase in proteoglycan production. We demonstrated that MMP12, MGP, and KRT19 are the major differential expressed genes between scoliotic and degenerated human NP cells. We found that MGP and MMP12 were significantly downregulated, while KRT19 expression was significantly upregulated, in NP cells treated by MSC-CM, especially CMSC-CM. The increased KRT19 expression in NP cells was also confirmed at protein level by confocal microscopy. Conclusion This is the first comparative study that discussed how different sources of MSCs affect the biological activities of cultured NP cells through paracrine effect. MGP2 and KRT193 has recently been reported to be associated with IVD degeneration. In this study, MSC-CM effectively upregulated KRT19 while downregulated MMP12 and MGP, suggesting that MSC-CM may promote a recovery of NP cell phenotype. In line with this finding is that MSC-CM treatment also enhanced overall cell metabolic activities, reduced apoptosis, and enhanced proteoglycan production of NP cells in culture. In all aspects tested, CMSC-CM showed stronger effect than BMSC-CM, suggesting that CMSC is a superior source of MSCs for future clinical application for IVD regeneration. Disclosure of Interest None declared References Wang L, Tran I, Seshareddy K, Weiss ML, Detamore MS. A comparison of human bone marrow-derived mesenchymal stem cells and human umbilical cord-derived mesenchymal stromal cells for cartilage tissue engineering. Tissue Eng Part A 2009;15(8):2259–2266 Lee CR, Sakai D, Nakai T, et al. A phenotypic comparison of intervertebral disc and articular cartilage cells in the rat. Eur Spine J 2007;16(12):2174–2185 Rutges J, Creemers LB, Dhert W, et al. Variations in gene and protein expression in human nucleus pulposus in comparison with annulus fibrosus and cartilage cells: potential associations with aging and degeneration. Osteoarthritis Cartilage 2010;18(3):416–423

Hif-αs Modulation of Sox9-Dependent Extracellular Matrix Production: Insight into Intervertebral Disc Cell Activity under Hypoxia

Introduction Intervertebral disc (IVD) degeneration leads to loss of mechanical function of motion segment and is attributed to deregulated extracellular matrix components in the nucleus pulposus (NP). The NP is rich in type II collagen (encoded by COL2A1) and aggrecan (encoded by AGC1), which are known to be regulated by the master chondrogenic transcription factor, Sox9.1 In situ studies of human degenerated NP illustrated a reduction of COL2A1 and absence of AGC1 expression, whereas SOX9 expression level remained high.2 This indicates that there may be other factors in the IVD that can regulate the Sox9-dependent matrix expression. Recent studies in aged rabbit IVD suggest that the abnormal NP cells may be contributed by a migration of the Sox9-expressing prechondrocytic cells from the annulus fibrosus (AF) adjacent to the endplate (EP).3, 4 Among the hypoxia inducible factor-α subunits (Hif-α), Hif-1α and Hif-2α (EPAS1) have been illustrated as important transcription factors in maintaining disc cell and matrix homeostasis, particularly in the hypoxic NP region.5-7 We hypothesized that Hif-αs may regulate the Sox9-dependent transcription of the extracellular matrix genes in response to IVD degeneration. First, we aimed at identification of Sox9 and Hif-αs co-expression in cells of non-degenerated and degenerated IVD. Second, we tested the modulatory effects of Hif-1α/Hif-2α on the expression of Col2a1 and Agc1 using Sox9-expressing mouse prechondrocytic cells as a model. Materials and Methods IVD were harvested from wild-type C57BL/6N mice at 3 and 6 month old. Disc degeneration was induced by an insertion of 25G needle into contralateral AF of 4-month old Lewis rat and discs were harvested post 2 to 4 weeks of operation. Lumbar IVD of scoliosis patients and degenerative disc disease patient were collected. All animal and human works were approved by local ethical committee. Comparative immunohistochemistry staining of Hif-1α, Hif-2α and Sox9 were studied in parrafilm sections of IVD. Luciferase-based promoter assays based on specific Col2a1 or Agc1 cis-acting elements via the overexpression for each of the HIF-α subunits and/or for Sox9 in ATDC5 cells were performed. Results Relative co-expression patterns of Hif-1α, Hif-2α and Sox9 were detected in AF, NP and EP of the mouse, rat and human IVD. Intracellular expression of Hif-1α, Hif-2α, and Sox9 were confirmed by immunofluorescence, indicating a possible role for Hif-αs-Sox9 tonic activity in the IVD. By luciferase-based promoter assay, Sox9 is demonstrated as a dominant transcription factor in the activation of transcription of Col2a1 and Agc1 in mouse chondroprogenitor cells. Overt additive transcriptional upregulation of Col2a1 and Agc1 was observed with a co-expression of HIF-1α and Sox9. Strikingly, Hif-2α inhibited the Sox9-dependent transcriptional upregulation of Col2a1 and Agc1. Conclusion Our findings indicate the presence of Hif-αs/Sox9 expressing cells in IVD. In addition, our data suggest that Hif-αs can modulate Sox9-mediated transcriptional regulation of Col2a1 and Agc1 in prechondrocytic cells, implicating a possible role of dynamic Hif-αs-Sox9 interaction in the IVD degeneration process. Further investigations of the orchestration mechanisms of Hif-αs/Sox9 in the IVD may provide insights in potential strategies to alleviate disc degeneration. Disclosure of Interest None declared References Richardson SM, Hoyland JA, Mobasheri R, Csaki C, Shakibaei M, Mobasheri A. Mesenchymal stem cells in regenerative medicine: opportunities and challenges for articular cartilage and intervertebral disc tissue engineering. J Cell Physiol 2010;222(1):23–32 Sive JI, Baird P, Jeziorsk M, Watkins A, Hoyland JA, Freemont AJ. Expression of chondrocyte markers by cells of normal and degenerate intervertebral discs. Mol Pathol 2002;55(2):91–97 Henriksson H, Thornemo M, Karlsson C, et al. Identification of cell proliferation zones, progenitor cells and a potential stem cell niche in the intervertebral disc region: a study in four species. Spine 2009;34(21):2278–2287 Henriksson HB, Svala E, Skioldebrand E, Lindahl A, Brisby H. Support of concept that migrating progenitor cells from stem cell niches contribute to normal regeneration of the adult mammal intervertebral disc: a descriptive study in the New Zealand white rabbit. Spine 2012;37(9):722–732 Rajpurohit R, Risbud MV, Ducheyne P, Vresilovic EJ, Shapiro IM. Phenotypic characteristics of the nucleus pulposus: expression of hypoxia inducing factor-1, glucose transporter-1 and MMP-2. Cell Tissue Res 2002;308(3):401–407 Risbud MV, Guttapalli A, Stokes DG, et al. Nucleus pulposus cells express HIF-1 alpha under normoxic culture conditions: a metabolic adaptation to the intervertebral disc microenvironment. J Cell Biochem 2006;98(1):152–159 Agrawal A, Gajghate S, Smith H, et al. Cited2 modulates hypoxia-inducible factor-dependent expression of vascular endothelial growth factor in nucleus pulposus cells of the rat intervertebral disc. Arthritis Rheum 2008;58(12):3798–3808

Cadherin 2 Is Required for Intervertebral Disc Homeostasis and Maintenance of Vacuolated Phenotype in Nucleus Pulposus Cells

Introduction Integrity of the nucleus pulposus (NP) has been implicated in the function and homeostasis of intervertebral disc (IVD). Understanding the regulation of NP cells would contribute to their engineering and development of therapeutics for treating IVD degeneration. Studies in mouse models indicate that early events of IVD degeneration involve segregation of the notochordal NP cell clusters,1 suggesting that disc degeneration may be associated with cell adhesion molecule activities. Cadherins are transmembrane glycoproteins that mediate calcium dependent cell adhesion.2 Based on microarray analysis, we have revealed specific expression of Cdh2 gene, encoding cadherin 2/N-cadherin, in rodent NP cells, suggesting a potential regulatory role of cadherins in IVD homeostasis. To date, the function of cadherin 2 in IVD and its relationship to IVD degeneration remain elusive. We hypothesize that cadherin 2 has a regulatory role in NP cells and that a deregulation of its activities has adverse effects on IVD homeostasis. We aimed to study the expression pattern of cadherin 2 in rodent discs during development, aging, and degeneration, and to investigate its function in the NP via a gene and protein ablation strategies. Materials and Methods Animal experiments were approved by local ethics committee. The vertebral columns of wild-type C57BL/6N mice were collected at different ages (n = 4): embryonic day (E) 12.5, E14.5, postnatal day 0 (P0), 3 months old, 6 months old, 1 year old, 1.5 and 2 years old. Progressive disc degeneration was induced by annulus puncture of 4-month-old inbred Lewis rats (n = 6) with 25G needle, and the discs were harvested after 2-, 4-, and 8-weeks of operation. Lumbar IVD of scoliosis patients and lower lumbar spine of the aborted fetus in the second trimester were used as controls. Immunohistochemistry was performed on paraffin sections to study the cadherin 2 expression pattern. For protein ablation study, 4-month-old inbred Lewis rats were anesthetized and the tail IVDs were exposed for injection of rabbit anti-cadherin 2 antibody or control rabbit IgG (n = 6) into the NP via 34G hypodermic needle. Cadherin 2 gene (Cdh2) was knocked out in the NP using notochord-specific Foxa2-Cre recombination strategy. Disc height was measured and expressed as disc height index (%DHI). The IVD were harvested by 2 and 8 weeks after operation (protein ablation) and from P0 and 1 month-old mutants (gene knockout) for histological analysis. Results By immunofluorescence, cadherin 2 was weakly detected in murine embryonic notochord and newborn NP. At 3- and 6-month old, strong cadherin 2 signals were specifically detected as foci along the cell-cell junctions of the vacuolated NP cells (notochordal cells). Annulus fibrosus showed no signals. In aged IVD, the notochordal cells were replaced by small chondrocyte-like cells with lower expression of cadherin 2. In puncture-induced degenerative rodent discs, the notochordal NP cells were replaced by rounded chondrocyte-like cells, showing reduced level of cadherin 2 expression. Human NP showed heterogeneous cadherin 2 expression. By injecting cadherin 2 antibody into rat NP to perturb its function in vivo, cadherin 2 expression was reduced along with reduction of disc height. Compared with the IgG injection control, cadherin 2 antibody ablation group showed a transformation of the notochordal NP cells into less vacuolated chondrocyte-like phenotype with upregulation of collagen II. Cdh2 conditional knockout mice (CKO) showed absence of cadherin 2 and a loss of vacuolated phenotype in the NP cells (Fig. 1A), displaying significantly smaller body size by 1-month old (Fig. 1B). Moreover, the mutant exhibited irregular annulus organization and reduced disc height in IVD (V). Conclusion Our study suggests cadherin 2 as a marker of notochordal NP cells. The lower expression of cadherin 2 in the aged and puncture-injured rodent IVD substantiates its association with IVD degeneration. The reduction of cadherin 2 positive NP cells in mature human IVD is consistent with gradual loss of notochordal cells after the first decade of life. Our parallel in vivo studies of gene/protein ablation support that cadherin 2 is essential to the maintenance of a vacuolated phenotype of the notochordal NP cells, and that a loss of cadherin 2 function may initiate degenerative changes in the IVD. In summary, our study implicates an important role of cadherin 2 in notochordal cell function and regulating IVD homeostasis. Acknowledgments This work is funded by the Area of Excellence grant (AoE/M-04/04) and the General Research Fund (HKU763712M) from the Research Grant Council of Hong Kong. Disclosure of Interest None declared References Yang F, Leung VY, Luk KD, Chan D, Cheung KM. Injury-induced sequential transformation of notochordal nucleus pulposus to chondrogenic and fibrocartilaginous phenotype in the mouse. J Pathol 2009;218(1):113–121 Leckband D, Prakasam A. Mechanism and dynamics of cadherin adhesion. Annu Rev Biomed Eng 2006;8:259–287

Lumbar Intervertebral Disc Transplantation: Healing and Remodeling of the Bony Structure

Introduction A previous human study suggested that intervertebral disc allograft transplantation in the cervical spine can relieve neurological symptoms and restore segmental kinematics. We have demonstrated that lumbar intervertebral disc transplantation could restore the global and segmental mobility after 12 months using a goat model. However, the healing process of the bone-bone interface and the subsequent remodeling of the bony endplate of the disc allograft are still unknown. This study examines this issue in a large animal model. Materials and Methods A total of 20 male goats between 6 and 9 months weighing between 17.5 and 25 kg were used in this study. Five goats served as disc allograft donors, and 15 goats as allograft recipients. Disc allograft transplantation without internal fixation was performed at lumbar L4/L5 using the retropsoas approach as previously described. Radiological healing was assessed with lateral radiographs of the lumbar spine at 1, 3, 6, 9, and 12 months posttransplantation. Five goats were sacrificed at 1.5, 6, and 12 months postoperatively, respectively. The transplanted segments together with the adjacent levels were then harvested en bloc and fixed in 10% formalin. Micro-CT scanning and three-dimensional reconstructions were conducted. Three volumes of interests (VOIs) were placed at the anterior, center, and posterior of the osteotomy sites; two VOIs were selected at the center of adjacent vertebral bodies near the epiphyseal line. Mean of the values at the three positions of osteotomy sites was calculated and compared with that of two adjacent vertebral VOIs as normal control to evaluate bone formation according to the following parameters: bone volume over total volume (BV/TV, %), bone surface density (BS/TV, 1/mm), trabecular thickness (Tb.Th, mm), trabecular number (Tb.N, 1/mm), trabecular separation (Tb.Sp, mm), trabecular pattern factor (Tb.Pf, 1/mm), structure model index (SMI), degree of anisotropy (DA), and connectivity density (Conn.Dn, 1/mm3). Sequentially, the sample was mid-sagittally cut. One half was decalcified for histological staining to morphologically observe new bone formation and bone remodeling; the other without decalcification was used for SEM and line-scan EDX analysis for assessing distribution profile of calcium (Ca) and phosphate (P) as well as the ratio of Ca/P at the newly formed bone of healing sites. Results Radiographically, increased radio-opacity was seen at the host-graft interface at 1 month postoperative, and bony union was seen after 3 months. Micro-CT images demonstrated more new bone formation with plate-like and well-connected architecture at the osteotomy sites at 1.5 months postoperative. These sites had higher BV/TV, BS/TV, Tb.N, and Conn.Dn, and had lower Tb.Pf and SMI than the mean of the control (p < 0.05). As the bone remodeling proceeds, the number (Tb.N), internal connection (Tb.Pf), and the structure (SMI, rod-like) of trabecular bone at the healing sites became similar with those of the control and remained unchanged until the final follow-up (p > 0.05). But the means of BV/TV, BS/TV, Tb.Th, and Conn.Dn were significantly greater than those of the control, while lower Tb.Sp at the healing site (p < 0.05). After 12 months, there was no difference between the healing site and the control in all 3D indices except the average of Conn.Dn (p < 0.01) and DA (p < 0.05). Despite the architecture of the remodeled trabecular bone was a little different than those from the control after 6 months, the profiles of Ca and P distribution as well as the ratio of Ca/P were comparable without significant difference. Histologically, resorption of the redundant vertebral bones and extensive new bone formation were noted in the healing site at 1.5 months; the subchondral bone and bony endplate of the disc allograft were still well preserved. In the form of creeping substitution, the subchondral bone was gradually replaced by dense and mature trabecular bone at 6 months postoperative. After continuous remodeling, the bony architecture became similar with that of the trabecular bone in the controls after 12 months. Conclusion Healing and remodeling of the host-graft bony interface was basically completed at 6 months posttransplantation. The bony structure of the disc allograft was replaced by newly formed trabecular bone through creeping substitution. Disclosure of Interest None declared Reference Ruan D, He Q, Ding Y, Hou L, Li J, Luk KD. Intervertebral disc transplantation in the treatment of degenerative spine disease: a preliminary study. Lancet 2007;369(9566):993–999

A Systematic Reevaluation of Enzymatic Isolation Methodology for Nucleus Pulposus Cells

Introduction The nucleus pulposus (NP) is the central part of IVD and understanding its function is essential for studying IVD degeneration. Enzymatic digestion by collagenase is used to release cells from NP tissues for cell phenotype characterization or further in vitro cell culture studies. For cell isolation from NP of different species, various protocols exist and vary greatly in terms of enzyme concentration, digestion duration, and type of precollagenase treatments such as digestion by pronase, which is a mixture of different proteases. In contrast to NP from other species, the cells in bovine or human mature NP have fewer cell-cell contacts and are surrounded by less gel-like matrix rich in proteoglycan and collagen. Thus we hypothesized that the pronase digestion step is not necessary to break the proteins which bind cells together in bovine or human NP. Bovine intervertebral disk (IVD) is an attractive candidate for studying IVD degeneration due to its anatomical and mechanical loading similarity with IVD in skeletally developed humans. And studies using bovine system can be more clinically relevant compared with other species. The avoidance of pronase treatment may better preserve the cell surface epitopes, which are critical for cell characterization recognizing surface protein markers. In this optimization study, we used bovine NP as a model to test different types of precollagenase treatments, different collagenase concentrations and different digestion durations to provide information about the optimal conditions for obtaining cells from bovine NP and human NP for different aims such as higher yield, higher viability, or reliable phenotyping. Materials and Methods Bovine NP tissue was isolated from bovine tail and digested by collagenase II with and without the prior digestion with pronase, trypsin, or dipase. The effect of collagenase concentration and digestion duration was studied by using two collagenase concentrations (0.05% vs 0.2%) and two incubation time points (4 to 8 hours vs. overnight). The number of extracted cells was estimated by counting with hematocytometer and the dead cells identified using Trypan blue stain. On the other hand, longer digestion duration and higher enzyme concentration would speed up the digestion but damages may be induced after prolonged digestion. Thus, the quality and phenotype of cells after prolonged digestion (with 0.5% collagenase for overnight digestion) plated on cell culture dishes was assessed using RT-qPCR after different cell passages. The mRNA levels of bovine NP cell markers (ACAN, COL2A1, CDH2, KRT18, KRT19) were monitored with GAPDH as the housekeeping gene and fresh bovine muscle as a control. Results Among the pre-collagenase enzyme treatments, pronase gives the highest cell yield. Longer duration of digestion or higher collagenase concentration without the use of pronase can achieve similar yield as with the pronase digestion step, indicating that the pronase digestion step may not be necessary. Overnight digestion may yield cells up to 7 times more than 4 hours of digestion. The majority of cells were viable and able to attach and grow on cell culture dishes though more dead cells were observed with overnight digestion and high enzyme concentration. High mRNA levels of NP markers were detected for the plated cells extracted with overnight digestion and high collagenase concentration (0.5%) compared with fresh muscle samples, indicating that the NP phenotype of cells was preserved. There is no significant difference in ACAN and COL2A1 mRNA expression in cells of the first three passages. On the other side, high quality of RNA can only be extracted from plated cells but not cells directly extracted out from the tissue after digestion with high enzyme concentration for overnight. This may be due to the unfavorable conditions for cells to synthesize mRNA during the collagen digestion, implying that different cell extraction protocols should be designed for cells extracted for different purposes. In short, pronase digestion may be omitted for isolation of cells from bovine NP tissue and this may better preserve the protein markers on the cell surface for characterization experiments. Overnight collagenase digestion gives higher cell yield and the majority of cells remain alive, although mRNA cannot be extracted immediately due to the harsh digestion conditions for the cells. Conclusion This study shows that higher cell yield can be obtained with longer digestion time and higher collagenase concentration though the cell viability may be sacrificed meanwhile. Pronase digestion may not be desirable for cell characterization using surface protein markers and shorter digestion duration seems preferable when high quality RNA is required for reliable RT-qPCR gene expression study. Due to the similarity of bovine NP and human NP, the systematic investigation of the digestion conditions using bovine NP in this study provides valuable information in extracting cells from both bovine NP and human NP for different purposes. This allows researchers to devise their extraction protocol based on a clear rationale, thus enabling more reliable results to be obtained in an efficient manner. I confirm having declared any potential conflict of interest for all authors listed on this abstract Yes Disclosure of Interest None declared

Effect of Cryopreservation on the Biomechanical Properties of the Intervertebral Discs

Intervertebral Disc (IVD) allograft transplantation is presented to be a practical treatment of severe Degenerative Disc Disease (DDD). Limited availability of fresh allograft tissues necessitate the establishment of tissue banking for long-term storage. However, the effects of the cryopreservants on the mechanical properties of the IVD remains unknown. We hypothesize that the appropriate use of cryopreservants can preserve the mechanical properties of the IVD. In this study porcine discs were cryopreserved in different cryopreservative agent (CPA) concentration following a stepwise freezing protocol. After four weeks of storage in liquid nitrogen, dynamic viscoelastic properties of the IVDs were tested using uniaxial compression at different frequencies. Compared to fresh IVDs, no significant differences were found in the elastic modulus (E’), viscous modulus (E”) and loss tangent (E”/E’) in the discs cryopreserved with 0-20% CPA. However, cryopreservation at 20% CPA mixture resulted in lower E’ and E”. Our data suggest properly cryopreserved IVD is able to maintain both elastic and viscous characteristics and distribute loads as of a normal IVD.