Fibrin-Hyaluronic Acid Hydrogel (RegenoGel) with Fibroblast Growth Factor-18 for In Vitro 3D Culture of Human and Bovine Nucleus Pulposus Cells.

We investigated the effects of a fibrin-hyaluronic acid hydrogel (FBG-HA) and fibroblast growth factor 18 (FGF-18) for nucleus pulposus (NP) regeneration. Healthy bovine (n = 4) and human degenerated NP cells (n = 4) were cultured for 14 days in FBG-HA hydrogel with FGF-18 (∆51-mutant or wild-type) in the culture medium. Gene expression, DNA content, and glycosaminoglycan (GAG) synthesis were evaluated on day 7 and 14. Additionally, histology was performed. Human NP cells cultured in FBG-HA hydrogel showed an increase in collagen type II (COL2) and carbonic anhydrase XII (CA12) gene expression after 14 or 7 days of culture, respectively. GAG release into the conditioned medium increased over 14 days. Healthy bovine NP cells showed increased gene expression of ACAN from day 7 to day 14. Wild type FGF-18 up-regulated CA12 gene expression of human NP cells. Histology revealed an increase of proteoglycan deposition upon FGF-18 stimulation in bovine but not in human NP cells. The FBG-HA hydrogel had a positive modulatory effect on human degenerated NP cells. Under the tested conditions, no significant effect of FGF-18 was observed on cell proliferation or GAG synthesis in human NP cells.

Opiates do not violate the viability and proliferative activity of human articular chondrocytes.

Articular cartilage injuries present a challenge for the clinician. Autologous chondrocyte implantation embedded in scaffolds are used to treat cartilage defects with favorable outcomes. Autologous serum is often used as a medium for chondrocyte cell culture during the proliferation phase of the process of such products. A previous report showed that opiate analgesics (fentanyl, alfentanil and diamorphine) in the sera have a significant inhibitory effect on chondrocyte proliferation. In order to determine if opiates in serum inhibit chondrocyte proliferation, twenty two patients who underwent knee arthroscopy and were anesthetized with either fentanyl or remifentanil were studied. Blood was drawn before and during opiate administration and up to 2 h after its discontinuation. The sera were used as medium for in vitro proliferation of both cryopreserved and freshly isolated chondrocytes, and the number and viability of cells were measured. There was no difference in the yield or cell viability between the serum samples of patients anesthetized with fentanyl when either fresh or cryopreserved human articular chondrocytes (hACs) were used. Some non-significant reduction in the yield of cells was observed in the serum samples of patients anesthetized with remifentanil when fresh hAC were used. We conclude that Fentanyl in human autologous serum does not inhibit in vitro hAC proliferation. Remifentanil may show minimal inhibitory effect on in vitro fresh hAC proliferation.

Nano-ghosts: Novel biomimetic nano-vesicles for the delivery of antisense oligonucleotides.

Antisense oligonucleotides (ASOs) carry an enormous therapeutic potential in different research areas, however, the lack of appropriate carriers for their delivery to the target tissues is hampering their clinical translation. The present study investigates the application of novel biomimetic nano-vesicles, Nano-Ghosts (NGs), for the delivery of ASOs to human mesenchymal stem cells (MSCs), using a microRNA inhibitor (antimiR) against miR-221 as proof-of-concept. The integration of this approach with a hyaluronic acid-fibrin (HA-FB) hydrogel scaffold is also studied, thus expanding the potential of NGs applications in regenerative medicine. The study shows robust antimiR encapsulation in the NGs using electroporation and the NGs ability to be internalized in MSCs and to deliver their cargo while avoiding endo-lysosomal degradation. This leads to rapid and strong knock-down of miR-221 in hMSCs in vitro, both in 2D and 3D hydrogel culture conditions (>90% and > 80% silencing efficiency, respectively). Finally, in vivo studies performed with an osteochondral defect model demonstrate the NGs ability to effectively deliver antimiR to endogenous cells. Altogether, these results prove that the NGs can operate as stand-alone system or as integrated platform in combination with scaffolds for the delivery of ASOs for a wide range of applications in drug delivery and regenerative medicine.

Mechanical Stress Inhibits Early Stages of Endogenous Cell Migration: A Pilot Study in an Ex Vivo Osteochondral Model.

Cell migration has a central role in osteochondral defect repair initiation and biomaterial-mediated regeneration. New advancements to reestablish tissue function include biomaterials and factors promoting cell recruitment, differentiation and tissue integration, but little is known about responses to mechanical stimuli. In the present pilot study, we tested the influence of extrinsic forces in combination with biomaterials releasing chemoattractant signals on cell migration. We used an ex vivo mechanically stimulated osteochondral defect explant filled with fibrin/hyaluronan hydrogel, in presence or absence of platelet-derived growth factor-BB or stromal cell-derived factor 1, to assess endogenous cell recruitment into the wound site. Periodic mechanical stress at early time point negatively influenced cell infiltration compared to unloaded samples, and the implementation of chemokines to increase cell migration was not efficient to overcome this negative effect. The gene expression at 15 days of culture indicated a marked downregulation of matrix metalloproteinase (MMP)13 and MMP3, a decrease of ß1 integrin and increased mRNA levels of actin in osteochondral samples exposed to complex load. This work using an ex vivo osteochondral mechanically stimulated advanced platform demonstrated that recurrent mechanical stress at early time points impeded cell migration into the hydrogel, providing a unique opportunity to improve our understanding on management of joint injury.

Enhanced chondrogenic phenotype of primary bovine articular chondrocytes in Fibrin-Hyaluronan hydrogel by multi-axial mechanical loading and FGF18.

Current treatments for cartilage lesions are often associated with fibrocartilage formation and donor site morbidity. Mechanical and biochemical stimuli play an important role in hyaline cartilage formation. Biocompatible scaffolds capable of transducing mechanical loads and delivering bioactive instructive factors may better support cartilage regeneration. In this study we aimed to test the interplay between mechanical and FGF-18 mediated biochemical signals on the proliferation and differentiation of primary bovine articular chondrocytes embedded in a chondro-conductive Fibrin-Hyaluronan (FB/HA) based hydrogel. Chondrocytes seeded in a Fibrin-HA hydrogel, with or without a chondro-inductive, FGFR3 selective FGF18 variant (FGF-18v) were loaded into a joint-mimicking bioreactor applying controlled, multi-axial movements, simulating the natural movements of articular joints. Samples were evaluated for DNA content, sulphated glycosaminoglycan (sGAG) accumulation, key chondrogenic gene expression markers and histology. Under moderate loading, samples produced particularly significant amounts of sGAG/DNA compared to unloaded controls. Interestingly there was no significant effect of FGF-18v on cartilage gene expression at rest. Following moderate multi-axial loading, FGF-18v upregulated the expression of Aggrecan (ACAN), Cartilage Oligomeric Matrix Protein (COMP), type II collagen (COL2) and Lubricin (PRG4). Moreover, the combination of load and FGF-18v, significantly downregulated Matrix Metalloproteinase-9 (MMP-9) and Matrix Metaloproteinase-13 (MMP-13), two of the most important factors contributing to joint destruction in OA. Biomimetic mechanical signals and FGF-18 may work in concert to support hyaline cartilage regeneration and repair. STATEMENT OF SIGNIFICANCE: Articular cartilage has very limited repair potential and focal cartilage lesions constitute a challenge for current standard clinical procedures. The aim of the present research was to explore novel procedures and constructs, based on biomaterials and biomechanical algorithms that can better mimic joints mechanical and biochemical stimulation to promote regeneration of damaged cartilage. Using a hydrogel-based platform for chondrocyte 3D culture revealed a synergy between mechanical forces and growth factors. Exploring the mechanisms underlying this mechano-biochemical interplay may enhance our understanding of cartilage remodeling and the development of new strategies for cartilage repair and regeneration.

A novel approach for knee osteoarthritis using high molecular weight hyaluronic acid conjugated to plasma fibrinogen - interim findings of a double-blind clinical study.

OBJECTIVE: Osteoarthritis (OA) is a widespread degenerative joint disease leading to progressive loss of function and pain. Available treatments do not provide long-term relief or improvement. This study aimed to assess the safety and efficacy of a novel intra articular supplement, made of high molecular-weight hyaluronic acid (HA) uniquely conjugated to either purified (RegenoGel) or autologous plasma-derived fibrinogen (RegenoGel-OSP), as a long-term treatment for knee OA. METHODS: Sixty-seven consecutive participants (mean age 67.26 ± 7 years) with symptomatic OA were randomly assigned to receive intraarticular injections of either RegenoGel, RegenoGel-OSP or saline solution (placebo). The active treatment groups received a second, repeat injection of the corresponding treatment at the 3-month evaluation, at which time, the placebo group was divided into two subgroups, one receiving RegenoGel and the other receiving RegenoGel-OSP. The OA symptoms were assessed by VAS, WOMAC, and IKDC questionnaires at baseline and at 1, 3, 4, and 6 months following the first injection. OA-related quality of life was evaluated by the SF-12 survey. RESULTS: Our preliminary data suggests that both fibrin-HA formulations have positive effects on OA symptoms for all assessed parameters with the most prominent trend for reduction in OA-associated pain. Pooled data analysis of RegenoGel and RegenoGel-OSP shows significantly improved VAS scores compared to placebo at three months after the first injection, and sustained for another three months after the second injection. Both RegenoGel, RegenoGel-OSP had an excellent safety profile. CONCLUSIONS: Interim analysis results indicate that RegenoGel and RegenoGel-OSP are safe and are potentially effective for at least six months in alleviating pain and symptoms of knee OA.

Fibroblast growth factor 4 and its novel splice isoform have opposing effects on the maintenance of human embryonic stem cell self-renewal.

Human embryonic stem cells (HESCs) are unique in their capacity to self-renew while remaining pluripotent. This undifferentiated state must be actively maintained by secreted factors. To identify autocrine factors that may support HESC growth, we have taken a global genetic approach. Microarray analysis identified fibroblast growth factor 4 (FGF4) as a prime candidate for autocrine signaling. Furthermore, the addition of recombinant FGF4 to HESCs supports their proliferation. We show that FGF4 is produced by multiple undifferentiated HESC lines, along with a novel fibroblast growth factor 4 splice isoform (FGF4si) that codes for the amino-terminal half of FGF4. Strikingly, although FGF4 supports the undifferentiated growth of HESCs, FGF4si effectively counters its effect. Furthermore, we show that FGF4si is an antagonist of FGF4, shutting down FGF4-induced Erk1/2 phosphorylation. Expression analysis shows that both isoforms are expressed in HESCs and early differentiated cells. However, whereas FGF4 ceases to be expressed in mature differentiated cells, FGF4si continues to be expressed after cell differentiation. Targeted knockdown of FGF4 using small interfering RNA increased differentiation of HESCs, demonstrating the importance of endogenous FGF4 signaling in maintaining their pluripotency. Taken together, these results suggest a growth-promoting role for FGF4 in HESCs and a putative feedback inhibition mechanism by a novel FGF4 splice isoform that may serve to promote differentiation at later stages of development.

Production of bioactive, post-translationally modified, heterotrimeric, human recombinant type-I collagen in transgenic tobacco.

Collagen's biocompatibility, biodegradability and low immunogenicity render it advantageous for extensive application in pharmaceutical or biotechnological disciplines. However, typical collagen extraction from animal or cadaver sources harbors risks including allergenicity and potential sample contamination with pathogens. In this work, two human genes encoding recombinant heterotrimeric collagen type I (rhCOL1) were successfully coexpressed in tobacco plants with the human prolyl-4-hydroxylase (P4H) and lysyl hydroxylase 3 (LH3) enzymes, responsible for key posttranslational modifications of collagen. Plants coexpressing all five vacuole-targeted proteins generated intact procollagen yields of approximately 2% of the extracted total soluble proteins. Plant-extracted rhCOL1 formed thermally stable triple helical structures and demonstrated biofunctionality similar to human tissue-derived collagen supporting binding and proliferation of adult peripheral blood-derived endothelial progenitor-like cells. Through a simple, safe and scalable method of rhCOL1 production and purification from tobacco plants, this work broadens the potential applications of human recombinant collagen in regenerative medicine.

Hydrogel-based delivery of antimiR-221 enhances cartilage regeneration by endogenous cells.

Articular cartilage is frequently injured by trauma or osteoarthritis, with limited and inadequate treatment options. We investigated a new strategy based on hydrogel-mediated delivery of a locked nucleic acid microRNA inhibitor targeting miR-221 (antimiR-221) to guide in situ cartilage repair by endogenous cells. First, we showed that transfection of antimiR-221 into human bone marrow-derived mesenchymal stromal cells (hMSCs) blocked miR-221 expression and enhanced chondrogenesis in vitro. Next, we loaded a fibrin/hyaluronan (FB/HA) hydrogel with antimiR-221 in combination or not with lipofectamine carrier. FB/HA strongly retained functional antimiR-221 over 14 days of in vitro culture, and provided a supportive environment for cell transfection, as validated by flow cytometry and qRT-PCR analysis. Seeding of hMSCs on the surface of antimiR-221 loaded FB/HA led to invasion of the hydrogel and miR-221 knockdown in situ within 7 days. Overall, the use of lipofectamine enhanced the potency of the system, with increased antimiR-221 retention and miR-221 silencing in infiltrating cells. Finally, FB/HA hydrogels were used to fill defects in osteochondral biopsies that were implanted subcutaneously in mice. FB/HA loaded with antimiR-221/lipofectamine significantly enhanced cartilage repair by endogenous cells, demonstrating the feasibility of our approach and the need to achieve highly effective in situ transfection. Our study provides new evidence on the treatment of focal cartilage injuries using controlled biomaterial-mediated delivery of antimicroRNA for in situ guided regeneration.

Polarizing receptor activation dissociates fibroblast growth factor 2 mediated inhibition of myelination from its neuroprotective potential.

Fibroblast growth factor (FGF) signaling contributes to failure of remyelination in multiple sclerosis, but targeting this therapeutically is complicated by its functional pleiotropy. We now identify FGF2 as a factor up-regulated by astrocytes in active inflammatory lesions that disrupts myelination via FGF receptor 2 (FGFR2) mediated activation of Wingless (Wnt) signaling; pharmacological inhibition of Wnt being sufficient to abrogate inhibition of myelination by FGF2 in tissue culture. Using a novel FGFR1-selective agonist (F2 V2) generated by deleting the N-terminal 26 amino acids of FGF2 we demonstrate polarizing signal transduction to favor FGFR1 abrogates FGF mediated inhibition of myelination but retains its ability to induce expression of pro-myelinating and immunomodulatory factors that include Cd93, Lif, Il11, Hbegf, Cxcl1 and Timp1. Our data provide new insights into the mechanistic basis of remyelination failure in MS and identify selective activation of FGFR1 as a novel strategy to induce a neuroprotective signaling environment in multiple sclerosis and other neurological diseases.

A placebo-controlled study comparing the efficacy of intra-articular injections of hyaluronic acid and a novel hyaluronic acid-platelet-rich plasma conjugate in a canine model of osteoarthritis.

BACKGROUND: The objective of this study was to assess the efficacy of intra-articular injections of hyaluronic acid (HA) and a novel, on-site conjugate of HA with autologous fibrinogen in platelet-rich plasma (HA-PRP) in a canine model of osteoarthritis (OA) METHODS: Twelve beagle dogs underwent a unilateral resection of the cranial cruciate ligament (CrCL) of the stifle joint. Clinical and radiographic signs of OA were confirmed in all dogs 8 weeks following CrCL resection and prior to treatment. The dogs were randomized into three groups: saline (n = 4), HA (n = 4), and HA-PRP (n = 4). Each dog received intra-articular injections of the respective substance into the affected joint at pre-determined time points. The dogs were assessed for adverse effects for 3 days after each injection and for lameness, pain, range of motion, kinetics, and radiographic OA severity prior to treatment and 3 months after injection. OA severity as determined by radiographic examination was not significantly different among the groups at any time point. The dogs were then humanely euthanatized and the stifle joint assessed by gross and histological examinations. RESULTS: Dogs treated with four weekly injections of HA or two biweekly injections of HA-PRP were significantly (p < 0.05) better than dogs treated with four weekly injections of saline at 2-, 4-, and 12-week time points based on a comfortable range of motion (CROM) and clinical lameness score. Gait analysis measuring symmetry and weight distribution on pressure sensor walkway showed significantly (p < 0.05) improved limb function for dogs treated with HA and HA-PRP compared with dogs treated with saline yet with better clinical outcome for the HA-PRP-treated group at 12 and 20 weeks follow-up. Gross and histological analysis of synovium and articular cartilage demonstrated significant (p < 0.05) improvement by both treatments groups compared to controls. There was however significantly (p < 0.05) less damage to the cartilage in the HA-PRP group compared to the HA-treated group. CONCLUSIONS: These data suggest that while injection of HA and HA-PRP may be sufficient for short-term amelioration of the symptoms associated with OA, treatment with HA-PRP conjugates may be superior, providing significantly better long-term cartilage preservation.

Fibrin-hyaluronic acid hydrogel-based delivery of antisense oligonucleotides for ADAMTS5 inhibition in co-delivered and resident joint cells in osteoarthritis.

To date no disease-modifying drugs for osteoarthritis (OA) are available, with treatment limited to the use of pain killers and prosthetic replacement. The ADAMTS (A Disintegrin and Metallo Proteinase with Thrombospondin Motifs) enzyme family is thought to be instrumental in the loss of proteoglycans during cartilage degeneration in OA, and their inhibition was shown to reverse osteoarthritic cartilage degeneration. Locked Nucleic Acid (LNA)-modified antisense oligonucleotides (gapmers) released from biomaterial scaffolds for specific and prolonged ADAMTS inhibition in co-delivered and resident chondrocytes, is an attractive therapeutic strategy. Here, a gapmer sequence identified from a gapmer screen showed 90% ADAMTS5 silencing in a monolayer culture of human OA chondrocytes. Incorporation of the gapmer in a fibrin-hyaluronic acid hydrogel exhibited a sustained release profile up to 14 days. Gapmers loaded in hydrogels were able to transfect both co-embedded chondrocytes and chondrocytes in a neighboring gapmer-free hydrogel, as demonstrated by flow cytometry and confocal microscopy. Efficient knockdown of ADAMTS5 was shown up to 14 days in both cell populations, i.e. the gapmer-loaded and gapmer-free hydrogel. This work demonstrates the use applicability of a hydrogel as a platform for combined local delivery of chondrocytes and an ADAMTS-targeting gapmer for catabolic gene modulation in OA.

Neutralizing antibody to FGFR2 can act as a selective biomarker and potential therapeutic agent for gastric cancer with FGFR2 amplification.

Fibroblast growth factor receptor 2 (FGFR2) amplification has been reported in 5-10% of gastric cancer (GC) and is associated with poor prognosis. In this study, we characterized the anti-tumor effect of PRO-007, a newly developed recombinant monoclonal antibody that targets FGFR2, in GC cell lines KATO III (with FGFR2 amplification) and NCI-N87 (without FGFR2 amplification). Validation was performed in parallel using two patient-derived tumor cells (PDCs) from patients with GC. Cell viability assays were performed using FGFR2-transfected NCI-N87 cells and FGFR2-knockdown KATO III cells that were generated using short hairpin RNA (shRNA). PRO-007 reduced KATO III cell viability (P = 0.0034) but not that of NCI-N87 cells (P = 0.3710). PRO-007 also significantly reduced KATO III cell invasiveness (P < 0.0001) but not NCI-N87 cell invasiveness (P = 0.8136). Immunoblot analysis showed that PRO-007 treatment decreased the levels of phosphorylated AKT and ERK. The FGFR2-inhibitory activity of PRO-007 was confirmed in genetically modified GC cell lines. Cell viability of FGFR2-overexpressing NCI-N87 cells was significantly decreased by PRO-007, while KATO III cells were significantly resistant to the treatment when FGFR2 was knocked down by FGFR2 shRNA transfection. Furthermore, PRO-007 had a synergistic effect with ramucirumab on the invasiveness of cancer cells with FGFR2 amplification. Consistent results were obtained using PDCs from patients with GC. Overall, these preclinical data support the further clinical development of PRO-007 as a potential therapeutic agent for patients with FGFR2-amplified GC.

P2Y receptors activated by diadenosine polyphosphates reestablish Ca(2+) transients in achondroplasic chondrocytes.

Achondroplasia is the most common type of dwarfism, characterised by a mutation in the gene that encodes the fibroblast growth factor receptor 3 (FGFR3). Achondroplasia mainly affects the chondrocytes and therefore bones do not grow properly since intracellular pathways are altered. In this sense, defective calcium signaling by mutant FGFR3 has been previously described. The purpose of this study was to investigate the presence of purinergic P2Y receptors and how the activation of these receptors can have influence on defective calcium signaling observed in achondroplasic chondrocytes. The presence of P2Y receptors was determined by immunocytochemical and western blot techniques. Calcium mobilization after stimulation with nucleotides, dinucleotides, or, FGF9 application, was measured using the ratiometric dye fura-2/AM and fluorescence imaging. Our results demonstrate the expression of P2Y(1), P2Y(2), P2Y(6) and P2Y(11) receptors in achondroplasic chondrocytes, as well as the activation of these receptors after nucleotides and dinucleotides exposure. The altered calcium signaling of achondroplasic chondrocytes was confirmed, since FGF9 treatment fails to induce calcium mobilization. However, achondroplasic chondrocytes pre-treated with Ap(4)A are able to respond with increases in intracellular calcium after FGF9 stimulation. These findings show the rescue effect of diadenosine tetraphosphate (Ap(4)A), acting by means of P2Y receptors, on defective calcium response triggered by achondroplasic FGFR3.

Effect of PPADS on achondroplasic chondrocytes: inhibition of FGF receptor type 3 over-activity.

Achondroplasia, results from a mutation in the FGF receptor type 3, leading to receptor hyperactivation and subsequent amplification of FGF receptor type 3 signals. We have tested the ability of pyridoxal-5'-phosphate-6-azophenyl-2', 4'-disulfonate (PPADS) to decrease the overactivation and signalling of FGF receptor type 3 in achondroplasic chondrocytes. PPADS reduced the tyrosine phosphorylation of FGF receptor type 3 triggered by fibroblast growth factor 9 (FGF9) (50% reduction), as well as the activation of extracellular signal-regulated kinases 1 and 2 (ERK1/2) pathway. As a consequence of this inhibitory effect on ERK1/2 activity the loss of extracellular matrix was also reversed by PPADS. The action of PPADS seems to be due to a mechanism independent of P2 receptor antagonism.

A mutation in a CD44 variant of inflammatory cells enhances the mitogenic interaction of FGF with its receptor.

Synovial fluid cells from joints of rheumatoid arthritis (RA) patients express a novel variant of CD44 (designated CD44vRA), encoding an extra trinucleotide (CAG) transcribed from intronic sequences flanking a variant exon. The CD44vRA mutant was detected in 23 out of 30 RA patients. CD44-negative Namalwa cells transfected with CD44vRA cDNA or with CD44v3-v10 (CD44vRA wild type) cDNA bound FGF-2 to an equal extent via their associated heparan sulfate chains. However, Namalwa cells, immobilizing FGF-2 via their cell surface CD44vRA, bound substantially more soluble FGF receptor-1 (FGFR-1) than did Namalwa cells immobilizing the same amount of FGF-2 via their cell surface CD44v3-v10. The former cells stimulated the proliferation of BaF-32 cells, bearing FGFR-1, more efficiently than did the latter cells. Finally, isolated primary synovial fluid cells from RA patients expressing CD44vRA bound more soluble FGFR-1 to their cell surface-associated FGF-2 than did corresponding synovial cells expressing CD44v3-v10 or synovial cells from osteoarthritis patients. The binding of soluble FGFR-1 to RA synovial cells could be specifically reduced by their preincubation with Ab's against the v3 exon product of CD44. Hence, FGF-2 attached to the heparan sulfate moiety expressed by the novel CD44 variant of RA synovium cells exhibits an augmented ability to stimulate FGFR-1-mediated activities. A similar mechanism may foster the destructive inflammatory cascade not only in RA, but also in other autoimmune diseases.

Effect of diadenosine polyphosphates in achondroplasic chondrocytes: inhibitory effect of Ap4A on FGF9 induced MAPK cascade.

Achondroplasia is characterised by a mutation in the gene that encodes for the FGF receptor type 3 (FGFR3), producing a hyperactivation of this receptor and a subsequent increase in MAPK activity. We have tested the ability of nucleotides to decrease the activation of MAPK in chondrocytes with achondroplasic FGFR3 receptor. Diadenosine tetraphosphate, Ap(4)A, reduced the phosphorylation of pERK1/2 triggered by FGF9 (38% reduction). Ap(4)A diminished the expression of achondroplasic FGFR3 receptor (65% reduction), stimulating FGFR3 receptor degradation. The action of Ap(4)A seems to be mediated by a dinucleotide receptor rather than by any other ATP receptor.

CD44 involvement in autoimmune inflammations: the lesson to be learned from CD44-targeting by antibody or from knockout mice.

CD44 is a multistructural and multifunctional glycoprotein, the diversity of which is generated by alternative splicing. In this communication we review some aspects related to CD44 structure and function in experimental autoimmune inflammation, focusing on research performed in our own laboratory. We have found that CD44 targeting by antibody, passively injected into DBA/1 mice with collagen-induced arthritis (CIA) and NOD mice with type I diabetes or actively generated by CD44 cDNA vaccination of SJL/j mice with autoimmune encephalomyelitis, markedly reduced the pathological manifestations of these diseases by attenuating cell migration of the inflammatory cells and/or by their apoptotic killing. However, genetic deletion of CD44 by knockout technology enhanced the development of CIA because of molecular redundancy mediated by RHAMM (a receptor of hyaluronan-mediated motility). The mechanisms that stand behind these findings are discussed.

Heterodimeric BMP-2/7 for nucleus pulposus regeneration-In vitro and ex vivo studies.

Intervertebral disc (IVD) degeneration is the leading trigger of low back pain, which causes disability and leads to enormous healthcare toll worldwide. Biological treatment with growth factors has evolved as potential therapy for IVD regeneration. Bone morphogenetic protein 2 (BMP-2) and BMP-7 have shown promise in this regard. In the current study, we evaluated the effect of BMP-2/7 heterodimer for disc regeneration both in vitro and in organ culture. Nucleus pulposus (NP) cells isolated from bovine caudal disc were cultured in a fibrin-hyaluronan (FBG-HA) hydrogel for up to 14 days. BMP-2/7 heterodimer covalently incorporated within the hydrogel up-regulated the aggrecan and type II collagen gene expression, and glycosaminoglycan synthesis of NP cells. The activity of the BMP-2/7 heterodimer was dose dependent. The higher dose of BMP-2/7 was further assessed in an IVD whole organ system. After 14 days of culture with cyclic dynamic load, the BMP-2/7 heterodimer delivered into the nucleotomized region showed potential to stimulate the gene expression and synthesis of proteoglycan in the remaining NP tissue after partial nucleotomy. The gene expression level of type I collagen and alkaline phosphatase in the native disc tissue were not affected by BMP-2/7 treatment, indicating no adverse fibroblastic or osteogenic effect on the disc tissue. Intradiscal delivery of BMP-2/7 heterodimer may be a promising therapeutic approach for NP regeneration. The current IVD whole organ partial nucleotomy model may be utilized for screening of other biomaterials or drugs to treat early degenerative disc disorders. © 2016 Orthopaedic Research Society. Published by Wiley Periodicals, Inc. J Orthop Res 35:51-60, 2017.

Distinct fibroblast growth factor (FGF)/FGF receptor signaling pairs initiate diverse cellular responses in the oligodendrocyte lineage.

Fibroblast growth factors (FGFs) have been implicated in numerous cellular processes, including proliferation, migration, differentiation, and survival. Whereas FGF-2, the prototypic ligand in a family of 22 members, activates all four tyrosine kinase FGF receptors (FGFR1-FGFR4), other members demonstrate a higher degree of selectivity. Oligodendrocytes (OLs), the myelin-producing cells of the CNS, are highly influenced by FGF-2 at all stages of their development. However, how other FGFs and their cognate receptors orchestrate the development of OLs is essentially undefined. Using a combination of specific FGF ligands and receptor blocking antibodies, we now show that FGF-8 and FGF-17 target OL progenitors, inhibiting their terminal differentiation via the activation of FGFR3, whereas FGF-9 specifically targets differentiated OLs, triggering increases in process growth via FGFR2 signaling; FGF-18 targets both OL progenitors and OLs via activation of both FGFR2 and FGFR3. These events are highly correlated with changes in FGF receptor expression from FGFR3 to FGFR2 as OL progenitors differentiate into mature OLs. In addition, we demonstrate that, although activation of FGFR1 by FGF-2 leads to proliferation of OL progenitors, it produces deleterious effects on differentiated OLs (i.e., aberrant reentry into cell cycle and down-regulation of myelin proteins with a loss of myelin membrane). These data suggest that ligand availability, coupled with changes in FGF receptor expression, yield a changing repertoire of ligand-receptor signaling complexes that contribute critically to the regulation of both normal OL development and potential OL/myelin pathogenesis.