Neocartilage integration in temporomandibular joint discs: physical and enzymatic methods

Integration of engineered musculoskeletal tissues with adjacent native tissues presents a significant challenge to the field. Specifically, the avascularity and low cellularity of cartilage elicit the need for additional efforts in improving integration of neocartilage within native cartilage. Self-assembled neocartilage holds significant potential in replacing degenerated cartilage, though its stabilization and integration in native cartilage require further efforts. Physical and enzymatic stabilization methods were investigated in an in vitro model for temporomandibular joint (TMJ) disc degeneration. First, in phase 1, suture, glue and press-fit constructs were compared in TMJ disc intermediate zone defects. In phase 1, suturing enhanced interfacial shear stiffness and strength immediately; after four weeks, a 15-fold increase in stiffness and a ninefold increase in strength persisted over press-fit. Neither suture nor glue significantly altered neocartilage properties. In phase 2, the effects of the enzymatic stabilization regimen composed of lysyl oxidase, CuSO₄ and hydroxylysine were investigated. A full factorial design was employed, carrying forward the best physical method from phase 1, suturing. Enzymatic stabilization significantly increased interfacial shear stiffness after eight weeks. Combined enzymatic stabilization and suturing led to a fourfold increase in shear stiffness and threefold increase in strength over press-fit. Histological analysis confirmed the presence of a collagen-rich interface. Enzymatic treatment additionally enhanced neocartilage mechanical properties, yielding a tensile modulus over 6 MPa and compressive instantaneous modulus over 1200 kPa at eight weeks. Suturing enhances stabilization of neocartilage, and enzymatic treatment enhances functional properties and integration of neocartilage in the TMJ disc. Methods developed here are applicable to other orthopaedic soft tissues, including knee meniscus and hyaline articular cartilage.     

Curing kinetics and mechanical properties of a composite hydrogel for the replacement of the nucleuspulposus

A polymer material system has been developed to propose an injectable, UV and insitu curable hydrogel with properties similar to the native nucleuspulposus of intervertebral disc. Neat hydrogels based on Tween® 20 trimethacrylates (T3) and N-vinyl-2-pyrrolidone (NVP) and composite hydrogels of same composition reinforced by nano-fibrillated cellulose were synthesized with different T3 concentrations and their curing kinetics was investigated by photorheology using UV light. The T3 concentration has an influence on the time of curing and final shear stiffness of the material. NFC does not alter the time of curing but increases the final mechanical performance of the hydrogels for a same chemical composition. Hydrogel samples, neat and composite, were then tested in unconfined compression at different hydration stages and in confined compression and their elastic modulus was determined. The amount of fluid present in the network is mostly responsible for the mechanical properties and NFC fibres proved to be an efficient reinforcement. The elastic modulus ranged from 0.02 to 8 MPa. Biocompatibility studies showed that cells are confluent at 90% and do not show any morphology change when in contact with the hydrogel. The present hydrogel can therefore be considered for NP replacement.     

<Emphasis Type="Italic">In vitro</Emphasis> properties of PLLA screws and novel bioabsorbable implant with elastic nucleus to replace intervertebral disc

The suitability of two different implant types for the replacement of the intervertebral disc was studied in vitro. Self-reinforced poly-L-lactide (SR-PLLA) screws Ø 4.5 mm were studied 24 weeks in vitro and cylindrical implants with elastic nucleus made of poly(L/D)lactide 96/4, poly(L/DL)lactide 70/30, Bioactive Glass n:o 13–93 and Polyactive^® 1000PEOT70PBT30 were studied 15 weeks in vitro. The cylindrical implant mimics the size and shape of the intervertebral disc. During the in vitro, there were no changes in compression properties with either implant types. The screws had sufficient modulus for intervertebral ossification in the canine model and the cylindrical implant showed also sufficient mechanical properties. These results suggest that both implant types could be used in clinical testing.     

Composite hydrogels for intervertebral disc prostheses

Different PHEMA/PCL semi-IPNs hydrogels and their relative composite systems reinforced with PET fibres have been investigated for potential use as intervertebral disc prostheses. Compression properties and water absorption were evaluated. Uniaxial compression tests on the swollen samples showed an increase of the modulus and maximum stress with increasing content of PCL and PET fibres. In particular, the composite PHEMA/PCL hydrogels showed compression properties similar to those expressed by the canine intervertebral discs in different spinal locations. The equilibrium water content of modified semi-IPNs decreased as function of the PCL and PET fibres. These tests indicate that the use of composite hydrogels as disc prostheses is very promising because it is possible to combine transport and mechanical properties which are crucial for the performance of the intervertebral disc.This paper was accepted for publication after the 1995 Conference of the European Society of Biomaterials, Oporto, Portugal, 10–13 September.     

Increase or decrease in stability after nucleotomy? Conflicting in vitro and in vivo results in the sheep model

Nucleotomy is a common surgical procedure to treat disc herniations. The potential occurrence of segmental instability after surgery, however, is suspected to necessitate re-operation and fusion. Although in vitro studies support the theory of destabilization after nucleotomy, a prior, in-house animal study contrarily revealed an increase in stability after surgery. To identify which structural compartment of the motion segment is decisive for increased stability after nucleotomy in vivo, the flexibilities of ovine motion segments were measured after different stepwise reductions at the anterior and posterior spinal column. Different test groups were used in which nucleotomy had been performed during surgery in vivo and under isolated in vitro conditions, respectively. In accordance with expectations, in vitro nucleotomy on ovine motion segments significantly increased flexibility. By contrast, nucleotomy significantly decreased flexibility 12 weeks after surgery. After removal of the posterior structures, however, the differences in flexibility diminished. The present results thus suggest that it might not exclusively be the trauma to the intervertebral disc during surgery which is decisive for post-operative stability, but rather adaptive mechanisms in the posterior structures. Therefore, care should be taken to minimize the damage to the posterior structures in the course of the surgical approach, which more likely compromises stability.     

Rapidly in situ forming adhesive hydrogel based on a PEG-maleimide modified polypeptide through Michael addition

Polyethylene glycol-maleimide modified ε-polylysine (EPL-PEG-MAL) with a unique comb-shaped structure was designed and used as a novel crosslinker for thiolated chitosan (CSS). Novel polysaccharide/polypeptide bionic hydrogels based on CSS and EPL-PEG-MAL could form rapidly in situ within 1 min via Michael addition under physiological conditions. Rheological studies showed that introduction of PEG can dramatically improve the storage modulus (G′) of the hydrogels and the optimal hydrogel system showed superior G′ of 1,614 Pa. The maximum adhesion strength reached 148 kPa, six times higher than that of fibrin glue. Cytotoxicity test indicated that the hydrogel is nontoxic toward growth of L929 cells. Gelation time, swelling ratio, storage modulus and adhesion strength of the hydrogels can be modulated by the content of PEG-maleimide, CSS concentration and molar ratio of maleimide group to thiol group. Benefiting from the fast gelation behaviors, desirable mechanical properties, relatively high adhesive performance and no cytotoxicity, these hydrogels have the potential applications as promising biomaterials for tissue adhesion and sealing.     

Replacing the nucleus pulposus of the intervertebral disk: prediction of suitable properties of a replacement material using finite element analysis

An axisymmetric finite element model of a human lumbar disk was developed to investigate the properties required of an implant to replace the nucleus pulposus. In the intact disk, the nucleus was modeled as a fluid, and the annulus as an elastic solid. The Young's modulus of the annulus was determined empirically by matching model predictions to experimental results. The model was checked for sensitivity to the input parameter values and found to give reasonable behavior. The model predicted that removal of the nucleus would change the response of the annulus to compression. This prediction was consistent with experimental results, thus validating the model. Implants to fill the cavity produced by nucleus removal were modeled as elastic solids. The Poisson's ratio was fixed at 0.49, and the Young's modulus was varied from 0.5 to 100 MPa. Two sizes of implant were considered: full size (filling the cavity) and small size (smaller than the cavity). The model predicted that a full size implant would reverse the changes to annulus behavior, but a smaller implant would not. By comparing the stress distribution in the annulus, the ideal Young's modulus was predicted to be approximately 3 MPa. These predictions have implications for current nucleus implant designs. © 2001 Kluwer Academic Publishers     

Intervertebral disc (IVD): Structure, degeneration, repair and regeneration

Low back pain affects a large portion of the population, resulting in high care costs for therapy and treatment. One primary cause of low back pain is the degeneration of the intervertebral disc (IVD) resulting in the compression of the spinal nerves and adjacent vertebrae. Exact causes of degeneration are unknown, but it is thought that natural aging, and both biological and genetic factors may play a significant role in the degenerative process. Conventional methods to alleviate low back pain include spinal fusion and artificial disc replacement. Traditional treatments through spinal fusion may eliminate pain yet do not restore disc function and lead to further degeneration of adjacent levels by altering disc biomechanics and natural kinematics. Recently, artificial IVD replacements have started to gain interest, with two IVD implants currently approved in the United States. Although these implants facilitate the preservation of motions and disc space height, they are unable to sustain compressive forces due to their lack of elasticity. In addition, the implants may produce wear debris that can cause osteolysis and other deleterious effects. As an alternative to these conventional approaches, tissue engineered IVD constructs offer the advantage of biointegration while preserving the essential attributes of natural motion and disc space restoration. There is a great need for the development of tissue engineered scaffolds that simulate the natural 3D morphology and microenvironment of the targeted tissue. Scaffolds should facilitate biological transport to satisfy nutrition and waste removal requirements within the IVD. The discrete tissue architectures of the nucleus pulposus (NP) and annulus fibrosus (AF) have posed great challenges to IVD tissue engineering. Current attempts have not been able to satisfy the biological functions and/or mechanical properties of native tissue. Therefore, these current scaffolds are far from satisfactory. This review highlights the challenges met when tissue engineering an IVD, while offering a perspective on which biomaterials and scaffold fabrication methods have the realistic ability to serve as scaffolding constructs for IVD regeneration.     

Characterization of injectable hydrogels based on poly(N-isopropylacrylamide)-g-chondroitin sulfate with adhesive properties for nucleus pulposus tissue engineering

The goal of this work is to develop an injectable nucleus pulposus (NP) tissue engineering scaffold with the ability to form an adhesive interface with surrounding disc tissue. A family of in situ forming hydrogels based on poly(N-isopropylacrylamide)-graft-chondroitin sulfate (PNIPAAm-g-CS) were evaluated for their mechanical properties, bioadhesive strength, and cytocompatibility. It was shown experimentally and computationally with the Neo-hookean hyperelastic model that increasing the crosslink density and decreasing the CS concentration increased mechanical properties at 37 °C, generating several hydrogel formulations with unconfined compressive modulus values similar to what has been reported for the native NP. The adhesive tensile strength of PNIPAAm increased significantly with CS incorporation (p < 0.05), ranging from 0.4 to 1 kPa. Live/Dead and XTT assay results indicate that the copolymer is not cytotoxic to human embryonic kidney (HEK) 293 cells. Taken together, these data indicate the potential of PNIPAAm-g-CS to function as a scaffold for NP regeneration.     

Human annulus fibrosus dynamic tensile modulus increases with degeneration

The annulus fibrosus (AF) of the intervertebral disc experiences cyclic tensile loading in vivo at various states of mechanical equilibrium. Disc degeneration is associated with alterations in the biochemical composition of the AF including decreased water content, decreased proteoglycan concentration, and increased collagen deposition that affect mechanical function of the AF in compression and shear. Such changes may also affect the dynamic viscoelastic properties of the AF and thus alter the disc’s ability to dissipate energy under physiologic loading. The objectives of this study were to quantify the dynamic viscoelastic properties of human AF in circumferential tension and to determine the effect of degeneration on these properties. Nondegenerate and degenerate human AF tensile samples were tested in uniaxial tension over a spectrum of loading frequencies spanning 0.01–2 Hz at several states of equilibrium strain to determine the dynamic viscoelastic properties (dynamic modulus, phase angle) using a linear viscoelastic model. The AF dynamic modulus increased at higher equilibrium strain levels. The AF behaved more elastically at higher frequencies with a decreased phase angle. Degeneration resulted in a higher dynamic modulus at all strain levels but had no effect on phase angle. The findings from this study elucidate the effect of degeneration on the dynamic viscoelastic properties of human AF and lend insight into the mechanical role of the AF in cyclic loading conditions.     

Evaluation of hydrogels for soft tissue adhesives <Emphasis Type="Italic">in vitro</Emphasis> and <Emphasis Type="Italic">in vivo</Emphasis> analyses

In this study, natural materials (sodium alginate, dextran, gelatin and carboxymethyl chitosan) were modified to get aldehyde components and amino components. Upon mixing the two-component solutions together, four kinds of Schiff base hydrogels formed successfully within 5-300 s and could seal the wound tissue. The cytotoxicity tests of hydrogel extraction solution confirmed that the hydrogels are nontoxic materials. The adhesive ability was evaluated in vivo by measuring the adhesive strength after sealing the skin incisions on the back of rats. All the hydrogels showed higher adhesive strength than that of commercial fibrin glue and the blank control. The histological staining observation by hematoxylin and eosin staining (HE) and Masson’s trichrome staining (MTC) methods suggested that the hydrogels had good biocompatibility and biodegradation in vivo. They have only normal initial inflammation to skin tissue and could improve the formation of new collagen in the incision section. So, the prepared hydrogels were both safe and effective tissue adhesive, which had the great potentials to be used as skin tissue adhesive.     

Microstructural and mechanical characteristics of PHEMA-based nanofibre-reinforced hydrogel under compression

Natural network-structured hydrogels (e.g. bacterial cellulose (BC)) can be synthesised with specific artificial hydrogels (e.g. poly(2-hydroxyethyl methacrylate) (PHEMA)) to form a tougher and stronger nanofibre-reinforced composite hydrogel, which possesses micro- and nano-porous structure. These synthetic hydrogels exhibit a number of advantages for biomedical applications, such as good biocompatibility and better permeability for molecules to pass through. In this paper, the mechanical properties of this nanofibre-reinforced hydrogel containing BC and PHEMA have been characterised in terms of their tangent modulus and fracture stress/strain by uniaxial compressive testing. Numerical simulations based on Mooney-Rivlin hyperelastic theory are also conducted to understand the internal stress distribution and possible failure of the nanofibre-reinforced hydrogel under compression. By comparing the mechanical characteristics of BC, PHEMA, and PHEMA-based nanofibre reinforced hydrogel (BC-PHEMA) under the compression, it is possible to develop a suitable scaffold for tissue engineering on the basis of fundamental understanding of mechanical and fracture behaviours of nanofibre-reinforced hydrogels.     

Design of Porous Alginate Hydrogels by Sacrificial CaCO3 Templates: Pore Formation Mechanism

Fabrication of porous alginate hydrogels with a well‐controlled architecture useful for tissue engineering is still a challenge. Here, CaCO₃‐based templating is utilized to design stable alginate gels with controlled pore dimensions in the range of 5–50 μm. The mechanism of pore formation is studied considering two factors affecting the pore size: i) osmotic pressure generated during the dissolution of sacrificial CaCO₃ templates and ii) alginate gel network density. Osmotic pressure can achieve an upper limit of 100 MPa but does not affect the gel porosity. Additional osmotic pressure (range of kPa) induced by dextrans pre‐encapsulated into CaCO₃ vaterite is also insufficient for pore enlargement. Pore stability depends merely on the gel network density and on the number of crosslinking calcium ions provided locally per unit time; pores are collapsed when template dissolution is too slow or if there is insufficient alginate concentration (below 2%). Young's modulus indicates the soft nature of the prepared hydrogels (tens of kPa) applicable as soft porous scaffolds with a tuned internal structure.     

Modulating the mechanical properties of photopolymerised polyethylene glycol–polypropylene glycol hydrogels for bone regeneration

Hydrogels formulated from single polymers are often insufficient in terms of their mechanical properties for use as bone substitute materials. Hence, hydrogels synthesised from combinations of polymers have been investigated to optimise the performance of such materials. In the current study, polypropylene glycol dimethacrylate was added to polyethylene glycol dimethacrylate of a variety of molecular weights and photopolymerised to form a series of hydrogels. Polyethylene glycol and polypropylene glycol have the same chemical structure with the exception of a methyl group on the later. Herein, the influence of the methyl group of polypropylene glycol on the mechanical properties of hydrogels for bone regeneration applications is reported. For both unconfined and cyclic compression testing, results demonstrated that the incorporation of PEGDMA into the precursor improves the compression strength of the hydrogels. For example, in unconfined compression tests the Young’s modulus varied between 6.62?±?0.31?MPa and 8.08?±?0.81?MPa with the incorporation of PEGDMA 400.     

Use of adipose stem cells and polylactide discs for tissue engineering of the temporomandibular joint disc

There is currently no suitable replacement for damaged temporomandibular joint (TMJ) discs after discectomy. In the present study, we fabricated bilayer biodegradable polylactide (PLA) discs comprising a non-woven mat of poly(L/D)lactide (P(L/D)LA) 96/4 and a P(L/DL)LA 70/30 membrane plate. The PLA disc was examined in combination with adipose stem cells (ASCs) for tissue engineering of the fibrocartilaginous TMJ disc in vitro. ASCs were cultured in parallel in control and chondrogenic medium for a maximum of six weeks. Relative expression of the genes, aggrecan, type I collagen and type II collagen present in the TMJ disc extracellular matrix increased in the ASC-seeded PLA discs in the chondrogenic medium. The hypertrophic marker, type X collagen, was moderately induced. Alcian blue staining showed accumulation of sulphated glycosaminoglycans. ASC differentiation in the PLA discs was close to that observed in pellet cultures. Comparison of the mRNA levels revealed that the degree of ASC differentiation was lower than that in TMJ disc-derived cells and tissue. The pellet format supported the phenotype of the TMJ disc-derived cells under chondrogenic conditions and also enhanced their hyalinization potential, which is considered part of the TMJ disc degeneration process. Accordingly, the combination of ASCs and PLA discs has potential for the development of a tissue-engineered TMJ disc replacement.     

Process Factors and Edgewise Compressive Properties of Scarf-repaired Honeycomb Sandwich Structures

Bonded repairs were conducted on flat and edge-closed composite sandwich panels that had undergone different levels of initial damage, and edgewise compression behaviors of repaired panel were tested. Experimental results indicate that these repair techniques can restore the compression performance of damaged panels effectively. The repaired specimens recovered an average of over 83 % of their strength. A k-sample Anderson-Darling test was used to analyze the influence of various parameters, including curing temperature, curing pressure, and repair configurations. After a thorough comparison, it was concluded that a high-temperature, high-pressure treatment can improve the mechanical performance of repaired panels, but the improvement is closely related to the structural complexity of the repaired region. A double-side repair scheme could be used to prevent the degradation of mechanical performance caused by the additional bending moment. The conclusions drawn in the present study provide further insight into the mechanical performance of repaired sandwich panels under edgewise compressive loads. These data facilitate the improved design methodology on bonded repair of composite sandwich structures.     

Bilayered HA/CS/PEGDA hydrogel with good biocompatibility and self-healing property for potential application in osteochondral defect repair

The fabrication of osteochondral tissue engineering scaffolds comprised of different layers is a big challenge. Herein, bilayers comprised of double network hydrogels with or without nano hydroxyapatite (HAp) were developed by exploiting the radical reaction of poly(ethylene glycol) diacrylate (PEGDA) and the Schiff-base reaction of N-carboxyethyl chitosan (CEC) and oxidized hyaluronic acid sodium (OHA) for osteochondral tissue engineering. The bilayered osteochondral scaffold was successfully fabricated based on the superior self-healing property of both hydrogels and evaluated by scanning electron microscopy, macroscopic observation and mechanical measurements. In addition, the hydrogels exhibited good biocompatibility as demonstrated by the in vitro cytotoxicity and in vivo implantation tests. The results indicated that the bilayered hydrogel had great potential for application in osteochondral tissue engineering.     

Characterization of porcine sclera using instrumented nanoindentation

Sclera tissue is the outermost protective layer of the eye and often involved in mechanical interactions during ocular medical procedures. In this study, instrumented nanoindentation is evaluated as a methodology for characterizing porcine sclera submerged in phosphate-buffered saline (PBS). Variances within a single sample and between samples are compared. Reduced modulus measurements are used to determine the effects of hydration, mounting conditions, and maximum applied load. Extended in vitro exposure beyond four hours to PBS is determined to decrease sample integrity. The use of glue to secure samples improved data consistency and had no negative effects on measurements. The data suggest that nanoindentation is a high precision method for measuring the reduced modulus of ultra low modulus soft tissue such as sclera (E_r ~ 30 kPa) and illustrates the importance of environmental factors upon material properties.     

Effect of composition on gelation kinetics of unfilled and nanoapatite-filled poly(lactide-ethylene oxide-fumarate) hydrogels

Oscillatory shear rheometry at small strains was used to investigate the effects of composition (concentration of macromer, crosslinker, initiator/accelerator, and filler) on the gel point and ultimate storage modulus of poly(l-lactide-co-ethylene oxide-co-fumarate) (PLEOF) hydrogels. PLEOF in situ polymerizing hydrogels can be used as biodegradable scaffolds for cell encapsulation and tissue regeneration. The time evolution of the viscoelastic properties of the polymerizing mixture during the sol-gel transition was monitored using mechanical rheometry. According to the results, while the ultimate storage modulus linearly increased with the concentration of methylene bisacrylamide (BISAM) crosslinker, the values of the storage and loss moduli at the gel point were insensitive to the concentration of BISAM. Similar behavior was observed for PLEOF hydrogels reinforced with hydroxyapatite nanoparticles.     

细菌纤维素/聚谷氨酸复合水凝胶的辐照制备及性能

采用Co~(60)-γ射线辐照交联法制备细菌纤维素/聚谷氨酸(BC/PGA)复合水凝胶。采用红外光谱和扫描电子显微镜等对复合水凝胶的结构进行表征,研究了BC引入对复合水凝胶的凝胶分数、热失重、溶胀性能、压缩性能和流变性能的影响,并利用CCK-8法对复合水凝胶进行了细胞毒性评价。研究结果表明,辐照作用下BC纳米纤维和PGA形成双交联复合凝胶网络,BC可有效增加复合水凝胶的压缩强度、储能模量(G')和凝胶分数,降低复合水凝胶的平衡溶胀度。50kGy辐照剂量下,相对于纯PGA水凝胶,复合水凝胶压缩强度增大5倍,G'增大10倍。同时复合水凝胶无细胞毒性,可安全应用于生物医学领域。