Streamlining quantitative joint-wide medial femoro-tibial histopathological scoring of mouse post-traumatic knee osteoarthritis models.

OBJECTIVES: Histological scoring remains the gold-standard for quantifying post-traumatic osteoarthritis (ptOA) in animal models, allowing concurrent evaluation of numerous joint tissues. Available systems require scoring multiple sections/joint making analysis laborious and expensive. We investigated if a single section allowed equivalent quantitation of pathology in different joint tissues and disease stages, in three ptOA models. METHOD: Male 10-12-week-old C57BL/6 mice underwent surgical medial-meniscal-destabilization, anterior-cruciate-ligament (ACL) transection, non-invasive-ACL-rupture, or served as sham-surgical, non-invasive-ACL-strain, or naïve/non-operated controls. Mice (n = 12/group) were harvested 1-, 4-, 8-, and 16-week post-intervention. Serial sagittal toluidine-blue/fast-green stained sections of the medial-femoro-tibial joint (n = 7/joint, 84 µm apart) underwent blinded scoring of 40 histology-outcomes. We evaluated agreement between single-slide versus entire slide-set maximum or median scores (weighted-kappa), and sensitivity/specificity of single-slide versus median/maximum to detect OA pathology. RESULTS: A single optimal mid-sagittal section showed excellent agreement with median (weighted-kappa 0.960) and maximum (weighted-kappa 0.926) scores. Agreement for individual histology-outcomes was high with only 19/240 median and 15/240 maximum scores having a weighted-kappa ≤0.4, the majority of these (16/19 and 11/15) in control groups. Statistically-significant histology-outcome differences between ptOA models and their controls detected with the entire slide-set were reliably reproduced using a single slide (sensitivity >93.15%, specificity >93.10%). The majority of false-negatives with single-slide scoring were meniscal and subchondral bone histology-outcomes (89%) and occurred in weeks 1-4 post-injury (84%). CONCLUSION: A single mid-sagittal slide reduced the time needed to score diverse histopathological changes by 87% without compromising the sensitivity or specificity of the analysis, across a variety of ptOA models and time-points.

Muscle spindles of the multifidus muscle undergo structural change after intervertebral disc degeneration.

PURPOSE: Proprioceptive deficits are common in low back pain. The multifidus muscle undergoes substantial structural change after back injury, but whether muscle spindles are affected is unclear. This study investigated whether muscle spindles of the multifidus muscle are changed by intervertebral disc (IVD) degeneration in a large animal model. METHODS: IVD degeneration was induced by partial thickness annulus fibrosus lesion to the L3-4 IVD in nine sheep. Multifidus muscle tissue at L4 was harvested at six months after lesion, and from six age-/sex-matched naïve control animals. Muscle spindles were identified in Van Gieson's-stained sections by morphology. The number, location and cross-sectional area (CSA) of spindles, the number, type and CSA of intrafusal fibers, and thickness of the spindle capsule were measured. Immunofluorescence assays examined Collagen I and III expression. RESULTS: Multifidus muscle spindles were located centrally in the muscle and generally near connective tissue. There were no differences in the number or location of muscle spindles after IVD degeneration and only changes in the CSA of nuclear chain fibers. The thickness of connective tissue surrounding the muscle spindle was increased as was the expression of Collagen I and III. CONCLUSION: Changes to the connective tissue and collagen expression of the muscle spindle capsule are likely to impact their mechanical properties. Changes in capsule stiffness may impact the transmission of length change to muscle spindles and thus transduction of sensory information. This change in muscle spindle structure may explain some of the proprioceptive deficits identified with low back pain.

Elevated hypertrophy, growth plate maturation, glycosaminoglycan deposition, and exostosis formation in the Hspg2 exon 3 null mouse intervertebral disc.

Heparan sulfate (HS) regulates diverse cell signalling events in intervertebral disc development and homeostasis. The aim of the present study was to investigate the effect of ablation of perlecan HS/CS on murine intervertebral disc development. Genetic models carrying mutations in genes encoding HS biosynthetic enzymes have identified multiple roles for HS in tissue homeostasis. In the present study, we utilised an Hspg2 exon 3 null HS/CS-deficient mouse to assess the role of perlecan HS in disc cell regulation. HS makes many important contributions to growth factor sequestration, stabilisation/delivery, and activation of receptors directing cellular proliferation, differentiation, and assembly of extracellular matrix. Perlecan HS/CS-mediated interactions promote extracellular matrix assembly/stabilisation and tissue functional properties, and thus, removal of perlecan HS/CS should affect extracellular matrix function and homeostasis. Hspg2 exon 3 null intervertebral discs accumulated significantly greater glycosaminoglycan in the nucleus pulposus, annulus fibrosus, and vertebral growth plates than C57BL/6 wild-type (WT) I intervertebral discs. Proliferation of intervertebral disc progenitor cells was significantly higher in Hspg2 exon 3 null intervertebral discs, and these cells became hypertrophic by 12 weeks of age and were prominent in the vertebral growth plates but had a disorganised organisation. C57BL/6 WT vertebral growth plates contained regular columnar growth plate chondrocytes. Exostosis-like, ectopic bone formation occurred in Hspg2 exon 3 null intervertebral discs, and differences were evident in disc cell maturation and in matrix deposition in this genotype, indicating that perlecan HS/CS chains had cell and matrix interactive properties which repressively maintained tissue homeostasis in the adult intervertebral disc.

Catabolism of Fibromodulin in Developmental Rudiment and Pathologic Articular Cartilage Demonstrates Novel Roles for MMP-13 and ADAMTS-4 in C-terminal Processing of SLRPs.

BACKGROUND: Cartilage regeneration requires a balance of anabolic and catabolic processes. AIM: To examine the susceptibility of fibromodulin (FMOD) and lumican (LUM) to degradation by MMP-13, ADAMTS-4 and ADAMTS-5, the three major degradative proteinases in articular cartilage, in cartilage development and in osteoarthritis (OA). METHODS: Immunolocalization of FMOD and LUM in fetal foot and adult knee cartilages using an FMOD matrix metalloprotease (MMP)-13 neoepitope antibody (TsYG11) and C-terminal anti-FMOD (PR184) and anti-LUM (PR353) antibodies. The in vitro digestion of knee cartilage with MMP-13, A Disintegrin and Metalloprotease with Thrompospondin motifs (ADAMTS)-4 and ADAMTS-5, to assess whether FMOD and LUM fragments observed in Western blots of total knee replacement specimens could be generated. Normal ovine articular cartilage explants were cultured with interleukin (IL)-1 and Oncostatin-M (OSM) ± PGE3162689, a broad spectrum MMP inhibitor, to assess FMOD, LUM and collagen degradation. RESULTS AND DISCUSSION: FMOD and LUM were immunolocalized in metatarsal and phalangeal fetal rudiment cartilages and growth plates. Antibody TsYG11 localized MMP-13-cleaved FMOD in the hypertrophic chondrocytes of the metatarsal growth plates. FMOD was more prominently localized in the superficial cartilage of normal and fibrillated zones in OA cartilage. TsYG11-positive FMOD was located deep in the cartilage samples. Ab TsYG11 identified FMOD fragmentation in Western blots of normal and fibrillated cartilage extracts and total knee replacement cartilage. The C-terminal anti-FMOD, Ab PR-184, failed to identify FMOD fragmentation due to C-terminal processing. The C-terminal LUM, Ab PR-353, identified three LUM fragments in OA cartilages. In vitro digestion of human knee cartilage with MMP-13, ADAMTS-4 and ADAMTS-5 generated FMOD fragments of 54, 45 and 32 kDa similar to in blots of OA cartilage; LUM was less susceptible to fragmentation. Ab PR-353 detected N-terminally processed LUM fragments of 39, 38 and 22 kDa in 65⁻80-year-old OA knee replacement cartilage. FMOD and LUM were differentially processed in MMP-13, ADAMTS-4 and ADAMTS-5 digestions. FMOD was susceptible to degradation by MMP-13, ADAMTS-4 and to a lesser extent by ADAMTS-5; however, LUM was not. MMP-13-cleaved FMOD in metatarsal and phalangeal fetal rudiment and growth plate cartilages suggested roles in skeletogenesis and OA pathogenesis. Explant cultures of ovine cartilage stimulated with IL-1/OSM ± PGE3162689 displayed GAG loss on day 5 due to ADAMTS activity. However, by day 12, the activation of proMMPs occurred as well as the degradation of FMOD and collagen. These changes were inhibited by PGE3162689, partly explaining the FMOD fragments seen in OA and the potential therapeutic utility of PGE3162689.

The adolescent idiopathic scoliotic IVD displays advanced aggrecanolysis and a glycosaminoglycan composition similar to that of aged human and ovine IVDs.

PURPOSE: The present study was designed to ascertain how altered biomechanics in adolescent idiopathic scoliotic (AIS) intervertebral discs (IVDs) affected tissue compositions and aggrecan processing compared to age matched and aged human IVDs. Newborn, 2- and 10-year-old ovine IVDs were also examined. METHODS: Aggrecan populations were separated by Sepharose CL2B chromatography, composite agarose polyacrylamide gel electrophoresis (CAPAGE) and identified by immunoblotting. The KS and CS content of IVD tissue extracts from AIS IVDs were compared with age-matched normal adolescent IVDs and with old human IVDs. Extracts from newborn, 2- and 10-year-old ovine IVDs were also examined in a similar manner. RESULTS: Adolescent idiopathic scoliotic IVD Aggrecan populations shared similar levels of polydispersity and aggregatability with hyaluronan as old IVD proteoglycans. CAPAGE demonstrated three aggrecan populations in AIS, aged human and ovine IVDs increased polydispersity and mobility in CAPAGE. AIS IVDs had GAG compositions similar to aged human and ovine IVDs. Sulphated KS (5-D-4) and chondroitin-6-sulphate, 3-B-3(+) were markers of tissue maturation, and chondroitin-4-sulphate, 2-B-6(+) was prominent in immature IVDs but its levels were lower in mature IVDs. DISCUSSION: Sulphated KS and 3-B-3(+) CS were prominently associated with IVD maturation and AIS IVDs, while the 2-B-6(+) CS isomer was associated with immature IVD tissues. The polydispersity of aggrecan in AIS IVDs, which was similar to in old human and ovine IVDs, reflected altered processing in the AIS IVDs in response to the biomechanical microenvironments the disc cells were exposed to in AIS IVDs. These slides can be retrieved under Electronic Supplementary Material.

Macrophage polarization contributes to local inflammation and structural change in the multifidus muscle after intervertebral disc injury.

PURPOSE: Intervertebral disk (IVD) lesion and its subsequent degeneration have a profound effect on the multifidus muscle. The subacute/early chronic phase of multifidus remodeling after IVD lesion has been proposed to be regulated by inflammatory processes. The balance between pro-inflammatory (M1) and anti-inflammatory (M2) macrophages plays an important role in maintaining tissue integrity after injury. The localization, polarization of macrophage subtypes and their mediation of the pro-inflammatory cytokine tumor necrosis factor (TNF) are unknown in paraspinal muscles during IVD degeneration. A sheep model of IVD degeneration was used to investigate the role of macrophages and TNF in the structural alterations that occur within the multifidus muscle. METHODS: Anterolateral lesions were induced at L3-4 IVD in sheep. Multifidus muscle tissue at L4 was harvested 3 and 6 months after lesion and used for immunofluorescence assays to examine total macrophage number, macrophage polarization between M1 and M2, and to assess the localization of TNF expression in muscle, adipose and connective tissues from injured and naïve control animals. RESULTS: A greater proportion of M1 macrophages is present in muscle at both 3 and 6 months after IVD lesion, and adipose tissue at 6 months. Total number of macrophages is unchanged. At 6 months, expression of TNF is increased in adipose and connective tissue and the proportion of TNF expressed by M1 macrophages is increased. CONCLUSIONS: These data support the proposal that macrophages and TNF (pro-inflammatory cytokine) play an active role in the subacute/early chronic phase of remodeling in muscle, adipose and connective tissues of the multifidus during IVD degeneration. This presents a novel target for treatment. These slides can be retrieved under Electronic Supplementary Material.

A Histopathological Scheme for the Quantitative Scoring of Intervertebral Disc Degeneration and the Therapeutic Utility of Adult Mesenchymal Stem Cells for Intervertebral Disc Regeneration.

The purpose of this study was to develop a quantitative histopathological scoring scheme to evaluate disc degeneration and regeneration using an ovine annular lesion model of experimental disc degeneration. Toluidine blue and Haematoxylin and Eosin (H&E) staining were used to evaluate cellular morphology: (i) disc structure/lesion morphology; (ii) proteoglycan depletion; (iii) cellular morphology; (iv) blood vessel in-growth; (v) cell influx into lesion; and (vi) cystic degeneration/chondroid metaplasia. Three study groups were examined: 5 × 5 mm lesion; 6 × 20 mm lesion; and 6 × 20 mm lesion plus mesenchymal stem cell (MSC) treatment. Lumbar intervertebral discs (IVDs) were scored under categories (i-vi) to provide a cumulative score, which underwent statistical analysis using STATA software. Focal proteoglycan depletion was associated with 5 × 5 mm annular rim lesions, bifurcations, annular delamellation, concentric and radial annular tears and an early influx of blood vessels and cells around remodeling lesions but the inner lesion did not heal. Similar features in 6 × 20 mm lesions occurred over a 3-6-month post operative period. MSCs induced a strong recovery in discal pathology with a reduction in cumulative histopathology degeneracy score from 15.2 to 2.7 (p = 0.001) over a three-month recovery period but no recovery in carrier injected discs.

Spatiotemporal Expression of 3-B-3(-) and 7-D-4 Chondroitin Sulfation, Tissue Remodeling, and Attempted Repair in an Ovine Model of Intervertebral Disc Degeneration.

OBJECTIVE: Examination of intervertebral disc (IVD) regeneration in an ovine annular lesion model. HYPOTHESIS: Sulfation motifs are important functional determinants in glycosaminoglycans (GAGs). Previous studies have correlated 3-B-3(-) and 7-D-4 chondroitin sulfate (CS) motifs in tissues undergoing morphogenetic transition in development. We hypothesize that these motifs may also be expressed in degenerate IVDs and may represent a reparative response. DESIGN: Induction of disc degeneration by 5 mm or 6 × 20 mm lesions in the annulus fibrosus (AF) over 6 or 3 to 6 months postoperation (PO). Tissue sections were stained with toluidine blue-fast green, 3-B-3(-) and 7-D-4 CS-sulfation motifs were immunolocalized in 3-month PO 6 × 20 mm lesion IVDs. Sulfated glycosaminoglycan (GAG), 3-B-3(-), and 7-D-4 epitopes were quantitated by ELISIA (enzyme-linked immunosorbent inhibition assay) in extracts of AF (lesion site and contralateral half) and nucleus pulposus (NP) 0, 3, and 6 months PO. RESULTS: Collagenous overgrowth of lesions occurred in the outer AF. Chondroid metaplasia in ~20% of the 6 × 20 mm affected discs resulted in integration of an outgrowth of NP tissue with the inner AF lamellae preventing propagation of the lesion. 3-B-3(-) and 7-D-4 CS sulfation motifs were immunolocalized in this chondroid tissue. ELISIA quantified CS sulfation motifs demonstrating an increase 3 to 6 months PO in the AF lesion and a reduction in sulfated GAG not evident in the contralateral AF. CONCLUSIONS: (1) Outer annular lesions underwent spontaneous repair. (2) Chondroid metaplasia of the inner 6 × 20 mm defect prevented its propagation suggesting an apparent reparative response.

The relationship between synovial inflammation, structural pathology, and pain in post-traumatic osteoarthritis: differential effect of stem cell and hyaluronan treatment.

BACKGROUND: Synovitis is implicated in the severity and progression of pain and structural pathology of osteoarthritis (OA). Increases in inflammatory or immune cell subpopulations including macrophages and lymphocytes have been reported in OA synovium, but how the particular subpopulations influence symptomatic or structural OA disease progression is unclear. Two therapies, hyaluronan (HA) and mesenchymal stem cells (MSCs), have demonstrated efficacy in some clinical settings: HA acting as device to improve joint function and provide pain relief, while MSCs may have immunomodulatory and disease-modifying effects. We used these agents to investigate whether changes in pain sensitization or structural damage were linked to modulation of the synovial inflammatory response in post-traumatic OA. METHODS: Skeletally mature C57BL6 male mice underwent medial-meniscal destabilisation (DMM) surgery followed by intra-articular injection of saline, a hyaluronan hexadecylamide derivative (Hymovis), bone marrow-derived stem cells (MSCs), or MSC + Hymovis. We quantified the progression of OA-related cartilage, subchondral bone and synovial histopathology, and associated pain sensitization (tactile allodynia). Synovial lymphocytes, monocyte/macrophages and their subpopulations were quantified by fluorescent-activated cell sorting (FACS), and the expression of key inflammatory mediators and catabolic enzyme genes quantified by real-time polymerase chain reaction (PCR). RESULTS: MSC but not Hymovis significantly reduced late-stage (12-week post-DMM) cartilage proteoglycan loss and structural damage. Allodynia was initially reduced by both treatments but significantly better at 8 and 12 weeks by Hymovis. Chondroprotection by MSCs was not associated with specific changes in synovial inflammatory cell populations but rather regulation of post-injury synovial Adamts4, Adamts5, Mmp3, and Mmp9 expression. Reduced acute post-injury allodynia with all treatments coincided with decreased synovial macrophage and T cell numbers, while longer-term effect on pain sensitization with Hymovis was associated with increased M2c macrophages. CONCLUSIONS: This therapeutic study in mice demonstrated a poor correlation between cartilage, bone or synovium (histo)pathology, and pain sensitization. Changes in the specific synovial inflammatory cell subpopulations may be associated with chronic OA pain sensitization, and a novel target for symptomatic treatment.

Intra-articular Treatment of Osteoarthritis with Diclofenac-Conjugated Polymer Reduces Inflammation and Pain.

The most common treatment for osteoarthritis is daily oral administration of a nonsteroidal anti-inflammatory drug such as diclofenac. This daily dosage regime is often associated with severe side effects. In this study, we explored the potential of utilizing a high molecular weight cross-linked polyurethane polymer covalently linked to diclofenac (C-DCF-PU) for intra-articular administration. We aim to exploit the advantages of local drug delivery by developing an implant with improved efficacy and reduced side effects. The polymer was synthesized from a diclofenac-functionalized monomer unit in a simple one-pot reaction, followed by cross-linking. In vitro drug release studies showed zero-order drug release for 4 days, followed by a gradual decline in drug release rate until diclofenac was depleted after 15 days. The cross-linked polymer was triturated to yield an injectable microgel formulation for administration. Whole animal fluorescence imaging of the rhodamine-labeled C-DCF-RH-PU showed good retention of the polymer in the knee joints of healthy rats, with approximately 30% of the injected dose still present 2 weeks post intra-articular administration. In a reactivation arthritis animal model, the C-DCF-RH-PU formulation reduced pain and significantly reduced inflammation after a short lag phase, showing that this drug delivery system warrants further development for long-term treatment of osteoarthritis with the benefit of reduced side effects.

Efficacy of administered mesenchymal stem cells in the initiation and co-ordination of repair processes by resident disc cells in an ovine (Ovis aries) large destabilizing lesion model of experimental disc degeneration.

BACKGROUND: Forty percent of low back pain cases are due to intervertebral disc degeneration (IVDD), with mesenchymal stem cells (MSCs) a reported treatment. We utilized an ovine IVDD model and intradiscal heterologous MSCs to determine therapeutic efficacy at different stages of IVDD. METHODOLOGY: Three nonoperated control (NOC) sheep were used for MSC isolation. In 36 sheep, 6 × 20 mm annular lesions were made at three spinal levels using customized blades/scalpel handles, and IVDD was allowed to develop for 4 weeks in the Early (EA) and late Acute (LA) groups, or 12 weeks in the chronic (EST) group. Lesion IVDs received injections of 10 × 106 MSCs or PBS, and after 8 (EA), 22 (LA) or 14 (EST) weeks recuperation the sheep were sacrificed. Longitudinal lateral radiographs were used to determine disc heights. IVD glycosaminoglycan (GAG) and hydroxyproline contents were quantified using established methods. An Instron materials testing machine and customized jigs analyzed IVD (range of motion, neutral zone [NZ] and stiffness) in flexion/extension, lateral bending and axial rotation. qRTPCR gene profiles of key anabolic and catabolic matrix molecules were undertaken. Toluidine blue and hematoxylin and eosin stained IVD sections were histopathologically scoring by two blinded observers. RESULTS: IVDD significantly reduced disc heights. MSC treatment restored 95% to 100% of disc height, maximally improved NZ and stiffness in flexion/extension and lateral bending in the EST group, restoring GAG levels. With IVDD qRTPCR demonstrated elevated catabolic gene expression (MMP2/3/9/13, ADAMTS4/5) in the PBS IVDs and expession normalization in MSC-treated IVDs. Histopathology degeneracy scores were close to levels of NOC IVDs in MSC IVDs but IVDD developed in PBS injected IVDs. DISCUSSION: Administered MSCs produced recovery in degenerate IVDs, restored disc height, composition, biomechanical properties, down regulated MMPs and fibrosis, strongly supporting the efficacy of MSCs for disc repair.

Achilles and tail tendons of perlecan exon 3 null heparan sulphate deficient mice display surprising improvement in tendon tensile properties and altered collagen fibril organisation compared to C57BL/6 wild type mice.

The aim of this study was to determine the role of the perlecan (Hspg2) heparan sulphate (HS) side chains on cell and matrix homeostasis in tail and Achilles tendons in 3 and 12 week old Hspg2 exon 3 null HS deficient (Hspg2Δ3 - ∕Δ3 -) and C57 BL/6 Wild Type (WT) mice. Perlecan has important cell regulatory and matrix organizational properties through HS mediated interactions with a range of growth factors and morphogens and with structural extracellular matrix glycoproteins which define tissue function and allow the resident cells to regulate tissue homeostasis. It was expected that ablation of the HS chains on perlecan would severely disrupt normal tendon organization and functional properties and it was envisaged that this study would better define the role of HS in normal tendon function and in tendon repair processes. Tail and Achilles tendons from each genotype were biomechanically tested (ultimate tensile stress (UTS), tensile modulus (TM)) and glycosaminoglycan (GAG) and collagen (hydroxyproline) compositional analyses were undertaken. Tenocytes were isolated from tail tendons from each mouse genotype and grown in monolayer culture. These cultures were undertaken in the presence of FGF-2 to assess the cell signaling properties of each genotype. Total RNA was isolated from 3-12 week old tail and Achilles tendons and qRT-PCR was undertaken to assess the expression of the following genes Vcan, Bgn, Dcn, Lum, Hspg2, Ltbp1, Ltbp2, Eln and Fbn1. Type VI collagen and perlecan were immunolocalised in tail tendon and collagen fibrils were imaged using transmission electron microscopy (TEM). FGF-2 stimulated tenocyte monolayers displayed elevated Adamts4, Mmp2, 3, 13 mRNA levels compared to WT mice. Non-stimulated tendon Col1A1, Vcan, Bgn, Dcn, Lum, Hspg2, Ltbp1, Ltbp2, Eln and Fbn1 mRNA levels showed no major differences between the two genotypes other than a decline with ageing while LTBP2 expression increased. Eln expression also declined to a greater extent in the perlecan exon 3 null mice (P < 0.05). Type VI collagen and perlecan were immunolocalised in tail tendon and collagen fibrils imaged using transmission electron microscopy (TEM). This indicated a more compact form of collagen localization in the perlecan exon 3 null mice. Collagen fibrils were also smaller by TEM, which may facilitate a more condensed fibril packing accounting for the superior UTS displayed by the perlecan exon 3 null mice. The amplified catabolic phenotype of Hspg2Δ3 - ∕Δ3 - mice may account for the age-dependent decline in GAG observed in tail tendon over 3 to 12 weeks. After Achilles tenotomy Hspg2Δ3 - ∕Δ3 - and WT mice had similar rates of recovery of UTS and TM over 12 weeks post operatively indicating that a deficiency of HS was not detrimental to tendon repair.

Ablation of Perlecan Domain 1 Heparan Sulfate Reduces Progressive Cartilage Degradation, Synovitis, and Osteophyte Size in a Preclinical Model of Posttraumatic Osteoarthritis.

OBJECTIVE: To investigate the role of the heparan sulfate (HS) proteoglycan perlecan (HSPG-2) in regulating fibroblast growth factor (FGF) activity, bone and joint growth, and the onset and progression of posttraumatic osteoarthritis (OA) in a mouse gene-knockout model. METHODS: Maturational changes were evaluated histologically in the knees of 3-, 6-, and 12-week-old wild-type (WT) mice and Hspg2(Δ3-/Δ3-) mice (Hspg2 lacking domain 1 HS, generated by ablation of exon 3 of perlecan). Cartilage damage, subchondral bone sclerosis, osteophytosis, and synovial inflammation were scored at 4 and 8 weeks after surgical induction of OA in WT and Hspg2(Δ3-/Δ3-) mice. Changes in cartilage expression of FGF-2, FGF-18, HSPG-2, FGF receptor 1 (FGFR-1), and FGFR-3 were examined immunohistochemically. Femoral head cartilage from both mouse genotypes was cultured in the presence or absence of interleukin-1α (IL-1α), FGF-2, and FGF-18, and the content and release of glycosaminoglycan (GAG) and expression of messenger RNA (mRNA) for key matrix molecules, enzymes, and inhibitors were quantified. RESULTS: No effect of perlecan HS ablation on growth plate or joint development was detected. After induction of OA, Hspg2(Δ3-/Δ3-) mice had significantly reduced cartilage erosion, osteophytosis, and synovitis. OA-induced loss of chondrocyte expression of FGF-2, FGF-18, and HSPG-2 occurred in both genotypes. Expression of FGFR-1 after OA induction was maintained in WT mice, while FGFR-3 loss after OA induction was significantly reduced in Hspg2(Δ3-/Δ3-) mice. There were no genotypic differences in GAG content or release between unstimulated control cartilage and IL-1α-stimulated cartilage. However, IL-1α-induced cartilage expression of Mmp3 mRNA was significantly reduced in Hspg2(Δ3-/Δ3-) mice. Cartilage GAG release in either the presence or absence of IL-1α was unaltered by FGF-2 in both genotypes. In cartilage cultures with FGF-18, IL-1α-stimulated GAG loss was significantly reduced only in Hspg2(Δ3-/Δ3-) mice, and this was associated with maintained expression of Fgfr3 mRNA and reduced expression of Mmp2/Mmp3 mRNA. CONCLUSION: Perlecan HS has significant roles in directing the development of posttraumatic OA, potentially via the alteration of FGF/HS/FGFR signaling. These data suggest that the chondroprotection conferred by perlecan HS ablation could be attributed, at least in part, to the preservation of FGFR-3 and increased FGF signaling.

Mesenchymal Stem Cell Treatment of Intervertebral Disc Lesion Prevents Fatty Infiltration and Fibrosis of the Multifidus Muscle, but not Cytokine and Muscle Fiber Changes.

STUDY DESIGN: Longitudinal case-control animal model. OBJECTIVE: To investigate effects of mesenchymal stem cell (MSC) treatment on multifidus muscle remodeling after intervertebral disc (IVD) lesion. SUMMARY OF BACKGROUND DATA: Lesion and degeneration of IVDs cause structural remodeling of the multifidus muscle. Proinflammatory cytokines are thought to contribute. MSC treatment restores IVD health after lesion but its effects on surrounding tissues remains unknown. Using an animal model of IVD degeneration, we assessed the effects of MSC treatment of IVDs on the structural remodeling and cytokine expression within the multifidus muscle. METHODS: An anterolateral lesion was performed on the L1-2, L3-4, and L5-6 IVDs in sheep. At either 4 (early treatment) or 12 (late treatment) weeks after IVD lesion, MSCs were injected into the lesioned IVD. Multifidus muscle was harvested from L2 (gene expression analysis) and L4 (histological analysis) at 3 or 6 months after IVD lesion and naïve controls for histological analysis of muscle, adipose, and connective tissue cross-sectional areas, and immunohistochemistry to study muscle fiber types. Real-time polymerase chain reactions quantified expression of tumor necrosis factor, interleukin-1ß, and transforming growth factor-ß1. RESULTS: MSC treatment of IVD lesion prevented the increased adipose and connective tissue cross-sectional area expected after IVD lesion. MSC treatment did not prevent slow-to-fast muscle fiber type transformation. Gene expression of proinflammatory cytokines within the muscle was altered by the MSC treatment of IVD. Increased interleukin-1ß expression was prevented in the early treatment group and tumor necrosis factor and transforming growth factor-ß1 expression was upregulated at 6 months. CONCLUSION: Results show that although MSC treatment prevents fatty infiltration and fibrosis of the multifidus muscle after IVD lesion, it cannot prevent a muscle inflammatory response and muscle fiber transformation. These findings highlight the potential role of MSC therapy after IVD injury, but reveals that other interventions may also be necessary to optimize recovery of muscle. LEVEL OF EVIDENCE: 4.

Allogeneic Mesenchymal Precursor Cells Promote Healing in Postero-lateral Annular Lesions and Improve Indices of Lumbar Intervertebral Disc Degeneration in an Ovine Model.

STUDY DESIGN: In-vivo ovine model of intervertebral disc degeneration (IVD) to evaluate treatment with stem cells. OBJECTIVE: To determine if stem cells delivered to the nucleus pulposus (NP) or the annulus fibrosus (AF) of degenerated lumbar IVDs leads to improved indices of disc health. SUMMARY OF BACKGROUND DATA: Previous studies assessing the efficacy of stem cell injections into degenerated IVDs have reported positive findings. However, studies have been limited to small animals, targeting solely the NP, with short term follow-up. METHODS: Mesenchymal precursor cells (MSC) were obtained from the iliac crest of 8-week-old sheep. IVD degeneration was induced by postero-lateral annulotomy at three lumbar levels in eight 2-year-old sheep. Six months later, each degenerated IVD was randomized to one of three treatments: Injection of MSC into (i) previously incised AF (AFI), (ii) NP (NPI), or (iii) no injection (negative control, NC). The adjacent IVD received injection of phosphate buffered saline into NP (positive control, PC). Radiographs and magnetic resonance imaging scans were obtained at baseline, 6, 9, and 12 months. Discs were harvested at 12 months for biochemical and histological analyses. RESULTS: IVD degeneration was consistently observed postannulotomy, and characterized by reduced disc height index (DHI), disc height (DH), glycosaminoglycan (GAG) content, and increased grade of disc degeneration.Six months after stem cell injection, DHI and DH had recovered in AFI and NPI groups when compared with NC group (P < 0.01). Mean Pfirrmann grade improved from 3.25 to 2.67 (AFI group) and from 2.96 to 2.43 (NPI group). Mean histopathological grade improved for both AFI (P < 0.002) and NPI (P < 0.02) groups. Both AFI and NPI groups demonstrated spontaneous repair of the postero-lateral annular lesion. CONCLUSION: In this large animal model, injection of MSCs into the annulus fibrosus or the nucleus pulposus of degenerated IVD resulted in significant improvements in disc health. LEVEL OF EVIDENCE: N/A.