Avner Yayon

Biomimetic fibrin-hyaluronan hydrogels for nucleus pulposus regeneration.

AIM To develop a biomimetic polymeric injectable hydrogel that can support nucleus pulposus (NP) regeneration. MATERIALS & METHODS Natural polymer-based hydrogels were synthesized using fibrinogen (FBG) and hyaluronic acid (HA), conjugated by a novel two-step procedure. Bovine NP cells were cultured in FBG-HA conjugate-based 3D beads in vitro and in a nucleotomized organ culture model. RESULTS FBG-HA conjugate-based hydrogels prepared with 235 KDa HA at a FBG/HA w/w ratio of 17:1 showed superior gel stability and mechanical properties and markedly increased glycosaminoglycan synthesis compared with a FBG/HA mixture-based hydrogels or fibrin gels. Gene-expression levels of NP markers were maintained in vitro. In organ culture, NP cells seeded in FBG-HA conjugate-based hydrogels showed better integration with native NP tissue compared with fibrin gels. Moreover, FBG-HA conjugate-based hydrogels restored compressive stiffness and disc height after nucleotomy under dynamic load. CONCLUSION Specific FBG-HA conjugate-based hydrogels may be suitable as injectable materials for minimally invasive, biological NP regeneration.

BMP-2 and BMP-2/7 Heterodimers Conjugated to a Fibrin/Hyaluronic Acid Hydrogel in a Large Animal Model of Mild Intervertebral Disc Degeneration.

Abstract Intervertebral disc (IVD) degeneration is etiologically associated with low back pain and is currently only treated in severe cases with spinal fusion. Regenerative medicine attempts to restore degenerated tissue by means of cells, hydrogels, and/or growth factors and can therefore be used to slow, halt, or reverse the degeneration of the IVD in a minimally invasive manner. Previously, the growth factors bone morphogenetic proteins 2 and 7 (BMP-2, -7) were shown to enhance disc regeneration, in vitro and in vivo. Since BMPs have only a short in vivo half-life, and to prevent heterotopic ossification, we evaluated the use of a slow release system for BMP-2 homodimers and BMP-2/7 heterodimers for IVD regeneration. BMP growth factors were conjugated to a fibrin/hyaluronic acid (FB/HA) hydrogel and intradiscally injected in a goat model of mild IVD degeneration to study safety and efficacy. Mild degeneration was induced in five lumbar discs of seven adult Dutch milk goats, by injections with the enzyme chondroitinase ABC. After 12 weeks, discs were treated with either FB/HA-hydrogel only or supplemented with 1 or 5 μg/mL of BMP-2 or BMP-2/7. BMPs were linked to the FB/HA hydrogels using a transglutaminase moiety, to be released through an incorporated plasmin cleavage site. After another 12 weeks, goats were sacrificed and discs were assessed using radiography, MRI T2* mapping, and biochemical and histological analyses. All animals maintained weight throughout the study and no heterotopic bone formation or other adverse effects were noted during follow-up. Radiographs showed significant disc height loss upon induction of mild degeneration. MRI T2* mapping showed strong and significant correlations with biochemistry and histology as shown before. Surprisingly, no differences could be demonstrated in any parameter between intervention groups. To our knowledge, this is the first large animal study evaluating BMPs conjugated to an FB/HA-hydrogel for the treatment of mild IVD degeneration. The conjugated BMP-2 and BMP-2/7 appeared safe, but no disc regeneration was observed. Possible explanations include too low dosages, short follow-up time, and/or insufficient release of the conjugated BMPs. These aspects should be addressed in future studies.

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.

Biomimetic Nucleus Pulposus Replacement for the Treatment of Degenerative Disc Disease

Introduction Nucleus pulposus (NP) replacement offers an alternative minimally invasive treatment for traditional spinal fusion or total disc replacement for degenerative disc disease (DDD). Recently, a novel polyurethane scaffold (PUS) with swelling capability in situ was developed to restore the mechanical functions of the intervertebral disc (IVD). In addition, a fibrinogen–hyaluronic acid (FBG–HA) conjugate-based hydrogel was manufactured to mimic the native NP extracellular matrix. The aim of this study is to evaluate the PUS and the FBG–HA hydrogel, with/without transglutaminase crosslinked bone morphogenetic protein (TG-BMP) 2/7 heterodimer in an organ culture system under dynamic load. Materials and Methods Discoid/ravioli-shaped polyurethane scaffolds were manufactured with a PU hydrogel-based core with swelling capacity in between two electrospun nanofiber envelope sheets. In addition, FBG–HA conjugate solution was synthesized with 235 kDa HA at FBG/HA w/w ratio of 17:1. FBG–HA hydrogels were prepared by mixing ⅔ volume of FBG–HA conjugate solution with ⅓ volume of thrombin solution (5.2 U/mL). Bovine IVDs with endplates were nucleotomized by incision through the endplate and refilled with either (1) PUS, (2) PUS surrounded by 50 to 80 µL FBG–HA or (3) PUS surrounded by 50 to 80µL FBG–HA containing 5,000 ng/mL TG–BMP 2/7. The endplate defect was closed with an endogenous endplate stopper and sealed with polymethyl methacrylate. Empty discs served as negative controls. To assess the mechanical compatibility of the different implants, dynamic compressive stiffness modulus (DCSM) was measured for each disc at different time points: intact, after nucleotomy, after refilling with biomaterial and free swelling recovery, after 3-hour dynamic load at 0 to 0.1 MPa, 0.1 Hz within a bioreactor system, and after free swelling recovery overnight. Moreover, biomaterials were evaluated in an organ culture system under dynamic load for 14 days at 0 to 0.1 MPa, 0.1 Hz for 3 hours/d. Disc height was recorded after load and recovery at day 1, 7, and 14. After 14 days, disc tissue was harvested and gene expression analyzed using real-time PCR. Glycosaminoglycan- (GAG) and collagen-content of the disc tissue was assessed and proteoglycan synthesis was analyzed by Sulfur-35 (35S) incorporation measurement. Histology was performed using Safranin O/Fast Green staining. One-way ANOVA was used to determine the statistical significance. Results After dynamic load, all three implant groups maintained their disc height, while it dropped by 7% in the empty controls (p < 0.001 vs. implant groups). The PUS and addition of FBG–HA hydrogel caused immediate restoration of DCSM (PUS: 73 ± 21%; p < 0.05 vs. empty control: 25 ± 5%). While addition of FBG–HA hydrogel surrounding the PUS delayed the stiffness restoring effect, stiffness also increased to 69 ± 16% after dynamic load and free swelling recovery (p < 0.05 vs. empty control: 27 ± 5%). GAG-/collagen-content was maintained in all three implant groups. Aggrecan and collagen-2 gene expression were upregulated in NP cells by TG-BMP2/7 and FBG–HA hydrogel, respectively, although not significantly. Conclusion Results indicate that PUS is able to restore the mechanical functions of nucleotomized discs. Addition of FBG–HA hydrogel does not affect the swelling capacity of PUS in situ. FBG–HA hydrogel and TG-BMP2/7 may further support the biological repair of NP tissue. Acknowledgment Funded by the European Commission under the FP7–NMP Project NPMimetic.

Nucleus Pulposus Replacement: Hydrogel or Scaffold?—An Organ Culture Study under Dynamic Load

Introduction Nucleus pulposus (NP) replacement offers a minimally invasive disc regeneration treatment alternative to traditional fusion or total disc replacement. An ideal NP replacement material should maintain motion and disc height, thus slowing down further disc degeneration.1 Various biomaterials have been investigated for their use as NP replacement, including hydrogel and nonhydrogel scaffolds, while no direct comparison study has been performed. Recently, a novel fibrin-hyaluronan (FBG-HA) conjugate hydrogel was developed to mimic native NP extracellular matrix. In addition, a novel polyurethane (PU) scaffold with swelling capability in situ was designed to restore native disc thickness and mechanical properties. The present study aimed to assess the FBG-HA hydrogel, the PU scaffold, and the combination of both in an organ culture system with dynamic load for the purpose of NP replacement. Materials and Methods FBG-HA conjugate solution was synthesized with 235 KDa HA at FBG/HA w/w ratio of 17:1 via a two-step procedure as described elsewhere.2 FBG-HA hydrogels were prepared by mixing two-thirds volume of FBG-HA conjugate solution with one-third volume of thrombin solution (5.2 U/mL) and allowed to polymerize at 37°C for 20 minutes. The PU scaffold was designed to have a flat discoid/ravioli shape that is composed of a based core with swelling capacity. Electrospinning nanofibers technology was used to manufacture an envelope sheet using a blend of polyurethane carbonates (ChronoFlex and HydroMed) that were dissolved in a mixture of dimethylformamide and dioxane. The envelope was cut to discs at diameter of 6 mm. The PU hydroMed was dissolved in 95% ethanol and dried to form a film. The film was cut to discs at diameter of 3 mm. One film disc was placed between two envelope discs and the circumference was heat welded to form a scaffold with ravioli shape. Caudal bovine intervertebral discs (IVDs) with endplates were obtained from calves (4-8 months) directly after slaughter. The discs were nucleotomized by incisions through the endplate. A 4-mm endplate core and the NP tissue below the endplate stopper were removed. The nucleotomized region was refilled with either (1) 50 to 80 µL FBG-HA hydrogel, (2) dry PU scaffold, or (3) PU scaffold surrounded by FBG-HA hydrogel. To close the defect, the removed endplate stopper was re-inserted, and the crack between the stopper and the remaining endplate was sealed by polymethyl methacrylate. Empty discs served as negative controls. To assess the mechanical compatibility of the different biomaterial implants, dynamic compressive stiffness modulus and disc height were measured for each disc at different time points: Intact, after nucleotomy, after refilling with biomaterial and free swelling culture overnight, after 3 hours dynamic load at 0 to 0.1 MPa, 0.1 Hz within a bioreactor system,3 and after free swelling recovery overnight. Results Dry PU scaffold was able to trap water and swell in situ already after 24 hours culture in the disc, as indicated by weight increase of 301 ± 33% and thickness increase of 103 ± 15%. Addition of FBG-HA hydrogel did not affect the swelling capability of the PU scaffold. After nucleotomy, the dynamic compressive stiffness modulus dropped to 30%. After refilling the NP cavity with PU scaffold and free swelling culture overnight, the disc stiffness modulus was restored to 73 ± 21% (p < 0.05 vs. control). After 3 hours dynamic load and overnight free swelling recovery, the stiffness modulus of PU scaffold refilled discs further increased to 82 ± 14% (p < 0.01 vs. control). Addition of FBG-HA hydrogel surrounding the PU scaffold delayed the stiffness restoration effect, while after 3 hours dynamic load and overnight free swelling recovery the stiffness also increased to 69 ± 16% (p < 0.05 vs. control) (Fig. 1A). After dissection and overnight culture, disc height increased by 2.6 to 4.5% due to swelling. Nucleotomy caused 3% decrease in disc height. Refilling the NP cavity with biomaterial did not change the disc height. However, after 3 hours dynamic load, all the three implant groups showed significantly higher disc height compared with empty discs (p < 0.001). After free swelling recovery overnight, the disc heights of all groups recovered (p < 0.001 implanted vs. control) (Fig. 1B). Conclusion Results indicate that PU scaffold may be a superior NP replacement material in terms of mechanical property repair compared with FBG-HA hydrogel. While combination of PU scaffold and FBG-HA hydrogel might result in better biological repair of NP, as FBG-HA hydrogel mimics native NP extracellular matrix and is a good carrier for cells implantation and/or drug delivery. Long-term study with the current well-established organ culture model will be performed to investigate the biological effect of PU scaffold, combined with FBG-HA hydrogel for NP replacement. Disclosure of Interest Z. Li: Conflict with the European Commission under the FP7 - NMP Project NPmimetic X. Chen: Conflict with the European Commission under the FP7 - NMP Project NPmimetic H. Sacks: Conflict with the European Commission under the FP7 - NMP Project NPmimetic; Conflict with Nicast Ltd A. Yayon: Conflict with the European Commission under the FP7 - NMP Project NPmimetic; Conflict with Procore Laboratories M. Alini: Conflict with the European Commission under the FP7 - NMP Project NPmimetic S. Grad: Conflict with the European Commission under the FP7 - NMP Project NPmimetic References Coric D, Mummaneni PV. Nucleus replacement technologies. J Neurosurg Spine 2008;8(2):115–120 Amit B, Barkay-Olami H, Yayon A. Water soluble reactive derivatives of carboxy polysaccharides and fibrinogen conjugates thereof. US patent 8,329,870, CIP US 13/685150 EP 2106263; India patent 4691/DELNP/2009 A. February 26, 2010 Jünger S, Gantenbein-Ritter B, Lezuo P, Alini M, Ferguson SJ, Ito K. Effect of limited nutrition on in situ intervertebral disc cells under simulated-physiological loading. Spine 2009;34(12):1264–1271

Gradient Organic Inorganic Nanocomposites for Tissue Repair at the Cartilage/Bone Interface

Damages to articular cartilage that are caused by trauma, age-related diseases (arthritis, arthrosis) and/or physical stress pose major medical problems. A possible solution is to introduce a biodegradable sponge-like scaffold containing cartilage-forming cells. In the current work we developed a model for a partially calcified functional biomedical membrane with a gradient of calcium phosphate crystal density to form the interface between bone and a sponge-like cell containing scaffold for cartilage regeneration. The membrane consists of a biocompatible, biodegradable, partially calcified hydrogel, in our case gelatin was used. One part is an organic-inorganic nanocomposite consisting of nanocrystalline calcium phosphate particles, formed in situ within the hydrogel, while the other part is the hydrogel without inorganic crystals. The experimental method used was one-dimensional single diffusion. Gelatin gels containing calcium or phosphate ions, respectively, were exposed from the upper side to a solution of the other constituent ion (i.e. a sodium phosphate solution was allowed to diffuse into a calcium containing gel and vice versa). Scanning electron microscopy (E-SEM), EDX, XRD and ATR-FTIR spectroscopy confirmed the existence within the gel of a density gradient of carbonate apatite crystals, with a dense top layer extending several microns into the gel. Ca/P atomic ratios were in the range characteristic of calcium deficient apatites. The effect of different experimental parameters on the calcification process within the gelatin membranes is discussed.