Biomaterials (v.27, #3)

Calendar (I).

Special Issue: Biomaterials for Spinal Applications by Lichun Lu; Michael J. Yaszemski (289).

Anterior spinal column augmentation with injectable bone cements by Jorrit-Jan Verlaan; F. Cumhur Oner; Wouter J.A. Dhert (290-301).
A vertebral fracture, whether originating from osteoporosis or trauma, can be the cause of pain, disability, deformation and neurological deficit. The treatment of vertebral compression fractures has, for many years until the advent of vertebroplasty, consisted of bedrest and analgesics. Vertebroplasty is a percutaneous technique during which bone cement is injected in a vertebral body to provide immediate pain relief by stabilization. Inflatable bone tamps can, prior to the injection of cement, be used to create a void in the vertebral body, in which case the technique is known as balloon vertebroplasty (or kyphoplasty). The chance of extracorporal cement leakage is smaller for balloon vertebroplasty than for vertebroplasty. Some authors also claim to have gained some correction in vertebral body height or angulation. Both interventions can be used for several indications, including osteoporotic compression fractures and osteolytic lesions of the vertebral body such as myeloma, hemangioma or metastasis, and also for traumatic burst fractures in combination with pedicle screw instrumentation. Polymethyl methacrylate cement is the bone void filler that is used most frequently, although the application of calcium phosphate cements has been studied widely in vitro, in vivo and also in small-scale clinical series. The clinical results of (balloon-) vertebroplasty are favorable with 85–95% of all patients experiencing immediate and long-lasting relief of pain. Serious complications are relatively rare but include neurological deficit and pulmonary embolism. In this paper, both vertebroplasty and balloon vertebroplasty and their respective indications, techniques and results are described in relation with the application and limitations of permanent and resorbable injectable bone cements.
Keywords: Thoracolumbar fracture; Vertebroplasty; Balloon vertebroplasty; Polymethyl methacrylate; Calcium phosphate; Anterior column augmentation;

A new in vivo screening model for posterior spinal bone formation: Comparison of ten calcium phosphate ceramic material treatments by Clayton E. Wilson; Moyo C. Kruyt; Joost D. de Bruijn; Clemens A. van Blitterswijk; F. Cumhur Oner; Abraham J. Verbout; Wouter J.A. Dhert (302-314).
This study presents a new screening model for evaluating the influence of multiple conditions on the initial process of bone formation in the posterior lumbar spine of a large animal. This model uses cages designed for placement on the decorticated transverse process of the goat lumbar spine. Five conduction channels per cage, each be defined by a different material treatment, are open to both the underlying bone and overlying soft tissue. The model was validated in ten adult Dutch milk goats, with each animal implanted with two cages containing a total of ten calcium phosphate material treatments according to a randomized complete block design. The ten calcium phosphate ceramic materials were created through a combination of material chemistry (BCP, TCP, HA), sintering temperature (low, medium, high), calcination and surface roughness treatments. To monitor the bone formation over time, fluorochrome markers were administered at 3, 5 and 7 weeks and the animals were sacrificed at 9 weeks after implantation. Bone formation in the conduction channels was investigated by histology and histomorphometry of non-decalcified sections using traditional light and epifluorescent microscopy. According to both observed and measured bone formation parameters, materials were ranked in order of increasing magnitude as follows: low sintering temperature BCP (rough and smooth)≈medium sintering temperature BCP≈TCP>calcined low sintering temperature HA>non-calcined low sintering temperature HA>high sintering temperature BCP (rough and smooth)>high sintering temperature HA (calcined and non-calcined). These results agree closely with those obtained in previous studies of osteoconduction and bioactivity of ceramics thereby validating the screening model presented in this study.
Keywords: Animal model; Bioactivity; Calcium phosphate; Osteoconduction; Osteogenesis;

In an effort to produce clinically relevant volumes of tissue-engineered bone products, we report a direct perfusion bioreactor system. Goat bone marrow stromal cells (GBMSCs) were dynamically seeded and proliferated in this system in relevant volumes (10 cc) of small sized macroporous biphasic calcium phosphate scaffolds (BCP, 2–6 mm). Cell load and cell distribution were shown using methylene blue block staining and 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) staining was used to demonstrate viability of the present cells. After 19 days of cultivation, the scaffolds were covered with a viable, homogeneous cell layer. The hybrid structures became interconnected and a dense layer of extracellular matrix was present as visualized by environmental scanning electron microscopy (ESEM). ESEM images showed within the extracellular matrix sphere like structures which were identified as calcium phosphate nodules by energy dispersive X-ray analysis (EDX). On line oxygen measurements during cultivation were correlated with proliferating GBMSCs. It was shown that the oxygen consumption can be used to estimate GBMSC population doubling times during growth in this bioreactor system. Implantation of hybrid constructs, which were proliferated dynamically, showed bone formation in nude mice after 6 weeks of implantation. On the basis of our results we conclude that a direct perfusion bioreactor system is capable of producing clinically relevant volumes of tissue-engineered bone in a bioreactor system which can be monitored on line during cultivation and show bone formation after implantation in nude mice.
Keywords: Bioreactor; Bone marrow cells; Flow perfusion; Online measurement; Bone tissue engineering; In vivo;

Polyetheretherketone as a biomaterial for spinal applications by Jeffrey M. Toth; Mei Wang; Bradley T. Estes; Jeffrey L. Scifert; Howard B. Seim; A. Simon Turner (324-334).
Threaded lumbar interbody spinal fusion devices (TIBFD) made from titanium have been reported to be 90% effective for single-level lumbar interbody fusion, although radiographic determination of fusion has been intensely debated in the literature. Using blinded radiographic, biomechanic, histologic, and statistical measures, we evaluated a radiolucent polyetheretherketone (PEEK)-threaded interbody fusion device packed with autograft or rhBMP-2 on an absorbable collagen sponge in 13 sheep at 6 months. Radiographic fusion, increased spinal level biomechanical stiffness, and histologic fusion were demonstrated for the PEEK cages filled with autograft or rhBMP-2 on a collagen sponge. No device degradation or wear debris was observed. Only mild chronic inflammation consisting of a few macrophages was observed in peri-implant tissues. Based on these results, the polymeric biomaterial PEEK may be a useful biomaterial for interbody fusion cages due to the polymer's increased radiolucency and decreased stiffness.
Keywords: Polyetheretherketone; Bone morphogenetic protein (BMP); PEEK; Spinal surgery; Spine fusion; Spine fusion cages;

Regenerative effects of transplanting mesenchymal stem cells embedded in atelocollagen to the degenerated intervertebral disc by Daisuke Sakai; Joji Mochida; Toru Iwashina; Akihiko Hiyama; Hiroko Omi; Masaaki Imai; Tomoko Nakai; Kiyoshi Ando; Tomomitsu Hotta (335-345).
Intervertebral disc (IVD) degeneration, a common cause of low back pain in humans, is a relentlessly progressive phenomenon with no currently available effective treatment. In an attempt to solve this dilemma, we transplanted autologous mesenchymal stem cells (MSCs) from bone marrow into a rabbit model of disc degeneration to determine if stem cells could repair degenerated IVDs. LacZ expressing MSCs were transplanted to rabbit L2–L3, L3–L4 and L4–L5 IVDs 2 weeks after induction of degeneration. Changes in disc height by plain radiograph, T2-weighted signal intensity in magnetic resonance imaging (MRI), histology, immunohistochemistry and matrix associated gene expressions were evaluated between normal controls (NC) without operations, sham operated with only disc degeneration being induced, and MSC-transplanted animals for a 24-week period.Results showed that after 24 weeks post-MSC transplantation, degenerated discs of MSC-transplanted group animals regained a disc height value of about 91%, MRI signal intensity of about 81%, compared to NC group discs. On the other hand, sham-operated group discs demonstrated the disc height value of about 67% and MRI signal intensity of about 60%. Macroscopic and histological evaluations confirmed relatively preserved nucleus with circular annulus structure in MSC-transplanted discs compared to indistinct structure seen in sham. Restoration of proteoglycan accumulation in MSC-transplanted discs was suggested from immunohistochemistry and gene expression analysis. These data indicate that transplantation of MSCs effectively led to regeneration of IVDs in a rabbit model of disc degeneration as suggested in our previous pilot study. MSCs may serve as a valuable resource in cell transplantation therapy for degenerative disc disease.
Keywords: Intervertebral disc; Nucleus pulposus; Cell therapy; Tissue engineering; Regenerative medicine; Mesenchymal stem cells; Stem cell; Atelocollagen;

Atelocollagen for culture of human nucleus pulposus cells forming nucleus pulposus-like tissue in vitro: Influence on the proliferation and proteoglycan production of HNPSV-1 cells by Daisuke Sakai; Joji Mochida; Toru Iwashina; Takuya Watanabe; Kaori Suyama; Kiyoshi Ando; Tomomitsu Hotta (346-353).
Nucleus pulposus (NP) is responsible for maintaining function and structure of the disc. Scaffolds to culture disc cells three-dimensionally are emphasized in recent reports on development of a new method for treating disc degeneration using cell transplantation and tissue engineering. Among artificial scaffolds and cell carrying materials, Atelocollagen is a collagen gel that has an advantage in safety issues over others. However, to date there has been no study that investigated culture of human nucleus pulposus cells in Atelocollagen. To investigate whether Atelocollagen could be used as a culture scaffold and if it has any effect on cell proliferation and proteoglycan (PG) production, as well as to find the optimal commercially available Atelocollagen for NP cell transplantation and tissue engineering, we cultured human NP cell line HNPSV-1, in three different Atelocollagen and compared with alginate. Furthermore, NP-like tissues were generated using these cells and different Atellocollagen solutions. Results showed that both DNA synthesis and content is significantly greater when cultured in Atelocollagen than in alginate. On the other hand, proteoglycan synthesis and accumulation was significantly greater in alginate compared with the 0.3% Atelocollagen scaffolds; with 3% Atelocollagen, however, results were similar. NP-like tissue generated by Atelocollagen showed good water and proteoglycan preservation. The current study demonstrates that the use of Atelocollagen as an in vitro culture scaffold for three-dimensional culture of human NP cell lines is indeed feasible and moreover, Atelocollagen possesses the potential to become a candidate scaffold for cell transplantation or tissue engineering for the treatment of intervertebral disc degeneration.
Keywords: Intervertebral disc; Disc degeneration; Nucleus pulposus; Atelocollagen; Tissue engineering; Scaffold;

Low-intensity pulsed ultrasound stimulates cell proliferation and proteoglycan production in rabbit intervertebral disc cells cultured in alginate by Toru Iwashina; Joji Mochida; Takeshi Miyazaki; Takuya Watanabe; Sadahiro Iwabuchi; Kiyoshi Ando; Tomomitsu Hotta; Daisuke Sakai (354-361).
Intervertebral disc degeneration, one of the major causes of low-back pain, is known to result from alteration in biosynthesis of proteoglycan in the disc. Therefore, upregulating the synthesis of proteoglycan in intervertebral disc cells may be one approach in treating disc degeneration. Based on the finding that low-intensity pulsed ultrasound stimulates proteoglycan synthesis in rat chondrocytes, we investigated whether low-intensity pulsed ultrasound stimulates biological properties of rabbit intervertebral disc cells in vitro. Nucleus pulposus cells and annulus fibrosus cells isolated from rabbits were cultured in alginate beads. Cells were stimulated for 20 min each day for 5–12 days, starting on the third day after seeding. An ultrasound signal consisting of a 200 μs burst sine wave of 0.5 MHz repeating at 1 kHz, with an intensity of 0, 7.5, 15, 30, 60, 120 mW/cm2 spatial and temporal average, was applied. DNA and proteoglycan synthesis were evaluated by measuring [3H]-thymidine and [35S]-sulfate incorporation. DNA and proteoglycan content in beads were measured by Hoechst 33258 dye method and dimethylmethylene blue assay. Results demonstrated positive effects on DNA synthesis and content, following low-intensity pulsed ultrasound stimulation with intensities of 7.5 and 15 mW/cm2. Furthermore, ultrasound stimulation significantly upregulated [35S]-sulfate incorporation and proteoglycan content compared to the control group, following 5 days of stimulation in both nucleus pulposus and annulus fibrosus cells. These findings suggest the possible application of low-intensity pulsed ultrasound in biological repair of intervertebral disc degeneration.
Keywords: Intervertebral disc; Disc degeneration; Ultrasound; Low-intensity pulsed ultrasound (LIPUS); Nucleus pulposus; Annulus fibrosus;

Biomechanical and biochemical characterization of composite tissue-engineered intervertebral discs by Hirokazu Mizuno; Amit K. Roy; Victor Zaporojan; Charles A. Vacanti; Minoru Ueda; Lawrence J. Bonassar (362-370).
Composite tissue-engineered intervertebral tissue was assembled in the shape of cylindrical disks composed of an outer shell of PGA mesh seeded with annulus fibrosus cells with an inner core of nucleus pulposus cells seeded into an alginate gel. Samples were implanted subcutaneously in athymic mice and retrieved at time points up to 16 weeks. At all retrieval times, samples maintained shape and contained regions of distinct tissue formation. Histology revealed progressive tissue formation with distinct morphological differences in tissue formation in regions seeded with annulus fibrosus and nucleus pulposus cells. Biochemical analysis indicated that DNA, proteoglycan, and collagen content in tissue-engineered discs increased with time, reaching >50% of the levels of native tissue by 16 weeks. The exception to this was the collagen content of the nucleus pulposus portion of the implants with were ∼15% of native values. The equilibrium modulus of tissue-engineered discs was 49.0±13.2 kPa at 16 weeks, which was between the measured values for the modulus of annulus fibrosus and nucleus pulposus. The hydraulic permeability of tissue-engineered discs was 5.1±1.7×10−14  m2/Pa at 16 weeks, which was between the measured values for the hydraulic permeability of annulus fibrosus and nucleus pulposus. These studies document the feasibility of creating composite tissue-engineered intevertebral disc implants with similar composition and mechanical properties to native tissue.
Keywords: Tissue engineering; Intervertebral disc; Annulus fibrosus; Nucleus pulposus; Biomechanics;

Three-dimensional culture of human disc cells within agarose or a collagen sponge: assessment of proteoglycan production by Helen E. Gruber; Gretchen L. Hoelscher; Kelly Leslie; Jane A. Ingram; Edward N. Hanley (371-376).
The objective of the present study was to assess proteoglycan production by human intervertebral disc cells cultured in vitro in selected cell carriers. Based on previous studies which evaluated disc cells seeded into collagen sponge, collagen gel, agarose, alginate or fibrin gel three-dimensional (3D) cell carriers, collagen sponge and agarose were found to provide superior microenvironments for formation of extracellular matrix (ECM). A standardized test design was used to evaluate ECM formed after 14 days of culture using the 1,9-dimethylmethylene blue (DMB) assay to assess sulfated glycosaminoglycan (S-GAG) production. Although agarose culture showed higher S-GAG levels compared to collagen sponge (2.94±2.20 (19) μg/ml S-GAG (mean±S.D. (n)) vs. 0.94±0.77 (22), respectively, p = 0.0003 ), this is off-set by the significantly lower proliferation rate associated with culture of disc cells in agarose.
Keywords: Disc; Tissue engineering; Proteoglycans; Extracellular matrix;

The use of tissue-engineering method holds great promise for treating degenerative disc disease [Gan JC, Ducheyne P, Vresilovic E, Shapiro IM. J Biomed Mater Res 2000; 51(4): 596–604]. This concept typically implies that nucleus pulposus (NP) cells are seeded on a scaffold, while the NP tissue is regenerated. Such hybrid implant is inserted into the host intervertebral disc. Because the success of a tissue engineering approach depends on maintenance or restoration of the mechanical function of the intervertebral disc, it is useful to study the initial mechanical performance of the disc after implantation of the hybrid. A three-dimensional finite element model (FEM) of the L2–L3 disc–vertebrae unit has been analyzed. The model took into account the material nonlinearities and it imposed different and complex loading conditions. In this study, we validated the model by comparison of its predictions with several sets of experimental data; we determined the optimal Young's modulus as well as the failure strength for the tissue-engineered scaffold under different loading conditions; and we analyzed the effects of implanted scaffold on the mechanical behavior of the intervertebral disc. The results of this study suggest that a well-designed tissue-engineered scaffold preferably has a modulus in the range of 5–10 MPa and a compressive strength exceeding 1.67 MPa. Implanted scaffolds with such properties can then achieve the goal of restoring the disc height and distributing stress under different loading conditions.
Keywords: Intervertebral disc; Finite element; Bioactive glass; Tissue engineering;

The potential of chitosan-based gels containing intervertebral disc cells for nucleus pulposus supplementation by Peter Roughley; Caroline Hoemann; Eric DesRosiers; Fackson Mwale; John Antoniou; Mauro Alini (388-396).
The suitability of chitosan-based hydrogels as scaffolds for the encapsulation of intervertebral disc (IVD) cells and the accumulation of a functional extracellular matrix mimicking that of the nucleus pulposus (NP) was investigated. The specific hypothesis under study was that the cationic chitosan would form an ideal environment in which large quantities of newly synthesized anionic proteoglycan could be entrapped. Indeed, all the formulations of cell-seeded chitosan hydrogels, studied under in vitro culture conditions, showed that the majority of proteoglycan produced by encapsulated NP cells was retained within the gel rather than released into the culture medium. This was not always the case when annulus fibrosus cells were encapsulated, as unlike the nucleus cells the annulus cells often did not survive when cultured in chitosan. The results support the concept that chitosan may be a suitable scaffold for cell-based supplementation to help restore the function of the NP during the early stages of IVD degeneration.
Keywords: Chitin/chitosan; Cell encapsulation; Hydrogel; Intervertebral disc; Nucleus pulposus; Proteoglycan;

Formation of a nucleus pulposus-cartilage endplate construct in vitro by Darla J. Hamilton; Cheryle A. Séguin; Jian Wang; Robert M. Pilliar; Rita A. Kandel (397-405).
Intervertebral disc (IVD) degeneration is a common problem and treatment options for persistent symptomatic disease are limited. Tissue engineering is being explored for its ability to reconstitute the functional components of the IVD. The purpose of this study was to determine whether it was possible to form in vitro a triphasic construct consisting of nucleus pulposus (NP), cartilage endplate (CEP), and a porous calcium polyphosphate (CPP) bone substitute. Bovine articular chondrocytes were placed on the top surface of a porous CPP construct and allowed to form cartilage in vitro. Nucleus pulposus cells were then placed onto the in vitro-formed hyaline cartilage. At 24 h scanning electron microscopy demonstrated that the NP cells maintained their rounded morphology, similar to NP cells placed directly on porous CPP. At 8 weeks histological examination of the triphasic constructs by light microscopy showed that a continuous layer of NP tissue had formed and was fused to the underlying cartilage tissue, which itself was integrated with the porous CPP. The incorporation of the cartilage layer was beneficial to the construct by improving tissue attachment to the CPP, as demonstrated by increased peak load and increased energy required for failure during shear loading when compared to a biphasic construct composed of nucleus pulposus-bone substitute only. This study demonstrates that it is possible to generate a multi-component construct with the incorporation of a CEP-like layer resulting in improved bone substitute-to-IVD tissue interface characteristics.
Keywords: Intervertebral disc; Nucleus pulposus; Cartilage endplate; Interface shear strength; Biomaterial;

Temperature-responsive hydroxybutyl chitosan for the culture of mesenchymal stem cells and intervertebral disk cells by Jiyoung M. Dang; Daniel D.N. Sun; Yoshitsune Shin-Ya; Ann N. Sieber; John P. Kostuik; Kam W. Leong (406-418).
Temperature-responsive polymers are attractive candidates for applications related to injectable delivery of biologically active therapeutics, such as stem cells. In this study, we evaluate the potential of thermosensitive hydroxybutyl chitosan (HBC) as a biomaterial for the culture of human mesenchymal stem cells (hMSC) and cells derived from the intervertebral disk, with the eventual goal of using the HBC polymer as an injectable matrix/cell therapeutic. Conjugation of hydroxybutyl groups to chitosan renders the polymer water soluble and thermally responsive. Below its lower critical solution temperature, a solution of HBC can be maintained indefinitely in its solvated state. Upon exposure to a 37 °C environment, within 60 s, a 3.8 wt% HBC solution rapidly forms a gel that can be maneuvered with forceps. Upon cooling, the gel once again is able to revert to its solvated state. The gel exhibits a dramatic increase in both G ′ and G ″ with increasing temperature, signifying a temperature-dependent enhancement of gel mechanical properties. Although a solid structure upon gelation, due to its physical nature of polymer interaction and gel formation, the gel exhibits a fluid-like viscoelastic behavior when exposed to shear stresses of up to 10% strain, with both G ′ and G ″ approaching zero with increasing shear stress. Formulations of HBC gels presented in this study have gelation temperatures ranging from 13.0 to 34.6 °C and water contents of 67–95%. Minimal cytotoxicity in MSC and disk cell cultures was observed with these polymers up to a concentration of 5 wt%. Detection of metabolic activity, genetic analysis of synthesized mRNA, and histological staining of MSC and disk cell cultures in these gels collectively indicate cell proliferation without a loss in metabolic activity and extracellular matrix production. This study suggests the potential of HBC gel as an injectable carrier for future applications of delivering therapeutics to encourage a biologically relevant reconstruction of the degenerated disk.
Keywords: Chitin/chitosan; Thermally responsive material; Hydrogel; Mesenchymal stem cells; Intervertebral disk;

Multiple-channel scaffolds to promote spinal cord axon regeneration by Michael J. Moore; Jonathan A. Friedman; Eric B. Lewellyn; Sara M. Mantila; Aaron J. Krych; Syed Ameenuddin; Andrew M. Knight; Lichun Lu; Bradford L. Currier; Robert J. Spinner; Richard W. Marsh; Anthony J. Windebank; Michael J. Yaszemski (419-429).
As molecular, cellular, and tissue-level treatments for spinal cord injury are discovered, it is likely that combinations of such treatments will be necessary to elicit functional recovery in animal models or patients. We describe multiple-channel, biodegradable scaffolds that serve as the basis for a model to investigate simultaneously the effects on axon regeneration of scaffold architecture, transplanted cells, and locally delivered molecular agents. Poly(lactic-co-glycolic acid) (PLGA) with copolymer ratio 85:15 was used for these initial experiments. Injection molding with rapid solvent evaporation resulted in scaffolds with a plurality of distinct channels running parallel along the length of the scaffolds. The feasibility of creating scaffolds with various channel sizes and geometries was demonstrated. Walls separating open channels were found to possess void fractions as high as 89%, with accessible void fractions as high as 90% through connections 220 μm or larger. Scaffolds degraded in vitro over a period of 30 weeks, over which time-sustained delivery of a surrogate drug was observed for 12 weeks. Primary neonatal Schwann cells were distributed in the channels of the scaffold and remained viable in tissue culture for at least 48 h. Schwann-cell containing scaffolds implanted into transected adult rat spinal cords contained regenerating axons at one month post-operation. Axon regeneration was demonstrated by three-dimensional reconstruction of serial histological sections.
Keywords: Nerve tissue engineering; Scaffold; Microstructure; Schwann cell; Drug release; Image analysis;

Freeze-dried poly(d,l-lactic acid) macroporous scaffold filled with a fibrin solution containing Schwann cells (SCs) lentivirally transduced to produce and secrete D15A, a bi-functional neurotrophin with brain-derived neurotrophic factor and neurotrophin-3 activity, and to express green fluorescent protein (GFP) were implanted in the completely transected adult rat thoracic spinal cord. Control rats were similarly injured and then implanted with scaffolds containing the fibrin solution with SCs lentivirally transduced to produce express GFP only or with the fibrin solution only. Transgene production and biological activity in vitro, SC survival within the scaffold in vitro and in vivo, scaffold integration, axonal regeneration and myelination, and hind limb motor function were analyzed at 1, 2, and 6 weeks after implantation. In vitro, lentivirally transduced SCs produced 87.5 ng/24 h/106  cells of D15A as measured by neurotrophin-3 activity in ELISA. The secreted D15A was biologically active as evidenced by its promotion of neurite outgrowth of dorsal root ganglion neurons in culture. In vitro, SCs expressing GFP were present in the scaffolds for up to 6 h, the end of a typical surgery session. Implantation of SC-seeded scaffolds caused modest loss of spinal nervous tissue. Reactive astrocytes and chondroitin sulfate glycosaminoglycans were present in spinal tissue adjacent to the scaffold. Vascularization of the scaffold was ongoing at 1 week post-implantation. There were no apparent differences in scaffold integration and blood vessel formation between groups. A decreasing number of implanted (GFP-positive) SCs were found within the scaffold during the first 3 days after implantation. Apoptosis was identified as one of the mechanisms of cell death. At 1 week and later time points after implantation, few of the implanted SCs were present in the scaffold. Neurofilament-positive axons were found in the scaffold. At 6 weeks post-grafting, myelinated axons were observed within and at the external surface of the scaffold. Axons did not grow from the scaffold into the caudal cord. All groups demonstrated a similar improvement of hind limb motor function. Our findings demonstrated that few seeded SCs survived in vivo, which could account for the modest axonal regeneration response into and across the scaffold. For the development of SC-seeded macroporous scaffolds that effectively promote axonal regeneration in the injured spinal cord, the survival and/or total number of SCs in the scaffold needs to be improved.
Keywords: CNS regeneration; Schwann cells; BDNF; NT-3; Poly (α-hydroxyacid); Biodegradable; Polymer; Spinal cord injury; Apoptosis; Caspase-3;

Although several approaches to stimulate axonal regeneration after spinal cord injury have succeeded in stimulating robust growth of axons into a lesion site, the growth is generally highly disorganized, losing the distinct arrangement of axonal tracts within the spinal cord. Previously described freeze-dried agarose scaffolds, composed of individual, uniaxial channels extending through their entire length, were prepared with and without recombinant Brain-Derived Neurotrophic Factor (BDNF) protein and tested in an adult rat model of spinal cord injury to determine whether regenerating axons could be guided across a site of injury in an organized fashion. After 1 month, both the cellular and axonal responses within and around scaffolds were evaluated. Scaffolds were found to be well integrated with host tissue, individual channels were penetrated by cells, and axons grew through scaffolds in a strikingly linear fashion. Furthermore, the regeneration was significantly augmented by the incorporation of BDNF protein into the walls and lumen of the scaffold. These findings clearly demonstrate that axonal regeneration can be organized and guided across a site of injury.
Keywords: Biopolymer; Scafold; Nerve regeneration; Spinal cord injury;

Stimulation of neurite outgrowth by neurotrophins delivered from degradable hydrogels by Jason A. Burdick; Matthew Ward; Ellen Liang; Michael J. Young; Robert Langer (452-459).
Degradable hydrogels are useful vehicles for the delivery of growth factors to promote the regeneration of diseased or damaged tissue. In the central nervous system, there are many instances where the delivery of neurotrophins has great potential in tissue repair, especially for treatment of spinal cord injury. In this work, hydrogels based on poly(ethylene glycol) that form via a photoinitiated polymerization were investigated for the delivery of neurotrophins. The release kinetics of these factors are controlled by changes in the network crosslinking density, which influences neurotrophin diffusion and subsequent release from the gels with total release times ranging from weeks to several months. The release and activity of one neurotrophic factor, ciliary-neurotrophic factor (CNTF), was assessed with a cell-based proliferation assay and an assay for neurite outgrowth from retinal explants. CNTF released from a degradable hydrogel above an explanted retina was able to stimulate outgrowth of a significantly higher number of neurites than controls without CNTF. Finally, unique microsphere/hydrogel composites were developed to simultaneously deliver multiple neurotrophins with individual release rates.
Keywords: Neurotrophins; Drug delivery; Biomaterials; Hydrogel;

Neurite bridging across micropatterned grooves by Joshua S. Goldner; Jan M. Bruder; Grace Li; Daniele Gazzola; Diane Hoffman-Kim (460-472).
After injury, regenerating axons must navigate complex, three-dimensional (3D) microenvironments. Topographic guidance of neurite outgrowth has been demonstrated in vitro with culture substrates that contain micropatterned features on the nanometer-micron scale. In this study we report the ability of microfabricated biomaterials to support neurite extension across micropatterned grooves with feature sizes on the order of tens of microns, sizes relevant to the design of biomaterials and tissue engineering scaffolds. Neonatal rat dorsal root ganglion (DRG) neurons were cultured on grooved substrates of poly(dimethyl siloxane) coated with poly-l-lysine and laminin. Here we describe an unusual capability of a subpopulation of DRG neurons to extend neurites that spanned across the grooves, with no underlying solid support. Multiple parameters influenced the formation of bridging neurites, with the highest numbers of bridges observed under the following experimental conditions: cell density of 125,000 cells per sample, groove depth of 50 μm, groove width of 30 μm, and plateau width of 200 μm. Bridges were formed as neurites extended from a neuron in a groove, contacted adjacent plateaus, pulled the neuron up to become suspended over the groove, and the soma translocated to the plateau. These studies are of interest to understanding cytoskeletal dynamics and designing biomaterials for 3D axon guidance.
Keywords: Micropatterning; Polydimethylsiloxane; Nerve tissue engineering; Laminin;

This paper describes a method for preparing substrates with micropatterns of positive guidance cues for the purpose of stimulating the growth of neurons. This method uses an oxidizing potential, applied to a micropattern of indium tin oxide in the presence of pyrrole and polyglutamic acid, to electrodeposit a matrix consisting of polypyrrole doped with polyglutamic acid. The resulting matrix subsequently can be modified with positive guidance cues via standard amide coupling reactions. Cells adhered to the micropatterned substrates can be stimulated electrically by the underlying electrodeposited matrix while they are in contact with positive guidance cues. This method can be extended to include both positive and negative guidance cues in a variety of combinations. To demonstrate the suitability of this method in the context of nerve guidance, dorsal root ganglia were grown in the presence of a micropatterned substrate whose surface was modified with molecules such as polylysine, laminin, or both. Cell adhesion and neurite extension were found to occur almost exclusively in areas where positive guidance cues were attached. This method is easy to execute and is of general utility for fundamental studies on the behavior of neurons in the presence of complex combinations of guidance cues as well as advanced bioelectronic devices such as neuronal networks.
Keywords: Electrochemistry; Laminin; Micropatterning; Nerve regeneration; Polypyrrole;

Characterization of non-neuronal elements within fibronectin mats implanted into the damaged adult rat spinal cord by V.R. King; J.B. Phillips; H. Hunt-Grubbe; R. Brown; J.V. Priestley (485-496).
Previous studies have shown that mats made from fibronectin (FN) integrate well into spinal cord lesion sites and support extensive axonal growth. Using immunohistochemistry, we have investigated the non-neuronal factors that contribute to these properties. Extensive vascularization was observed in FN mats by 1 week along with heavy macrophage infiltration by 3 days post-implantation. By 1 week post-implantation, laminin tubules had formed and were associated with axons and p75 immunoreactive Schwann cells. By 4 weeks post-implantation, most axons were associated with Schwann cell derived myelin. Few oligodendrocytes were present within the mat, even with an increase in the number of oligodendrocyte precursors around the implant site by 7 days post-implantation. Astrocyte proliferation also occurred in the intact tissue, with a prominent glial scar forming around the implant within 4 weeks. However, by 2 months post-implantation astrocytes were present in the FN implant site and were intermingled with the axons. Axonal ingrowth and integration of the FN mats is probably due to the ability of FN mats to support and organize infiltration of Schwann cells and deposition of laminin. At later time points, myelinated axons remain in the implant site, even after other elements (e.g. macrophages and laminin) have disappeared. Both of these properties are likely to be important in the design of biomaterial bridges for CNS regeneration.
Keywords: Fibronectin; Nerve tissue engineering; Laminin; Macrophage;

In situ gelling hydrogels for conformal repair of spinal cord defects, and local delivery of BDNF after spinal cord injury by Anjana Jain; Young-Tae Kim; Robert J. McKeon; Ravi V. Bellamkonda (497-504).
Permanent functional loss usually occurs after injury to the spinal cord. Currently, a clinical strategy to promote regeneration in the injured spinal cord does not exist. It has become evident that in order to promote regeneration, a growth permissive substrate at the injury site is critical. In this study, we report the utilization of an agarose scaffold that gels in situ, conformally filling an irregular, dorsal over-hemisection spinal cord defect in adult rats. Besides being growth permissive, the scaffolds also serve as carriers of trophic factors when embedded with BDNF releasing microtubules. We report that our thermo-reversible scaffolds are capable of supporting 3D neurite extension in vivo and are effective carriers of drug delivery vehicles for sustained local delivery of trophic factors. We demonstrate that BDNF encourages neurite growth into the scaffolds, and reduces further the minimal inflammatory response agarose gels generate in vivo as evidenced by quantitative analysis of the extent of NF-160 kDA positive neurons and axons, GFAP positive reactive astrocytes, and CS-56 positive chondroitin sulfate proteoglycan at the interface of the scaffold and host spinal cord. We suggest that these thermo-reversible scaffolds have great potential to serve as growth permissive 3D scaffolds, and to present neurotrophic factors and potentially anti-scar agents to the injury site and enhance regeneration after spinal cord injury.
Keywords: In situ gelling hydrogel; Spinal cord regeneration; BDNF delivery; CNS drug delivery; CNS scaffolds;

Coil-reinforced hydrogel tubes promote nerve regeneration equivalent to that of nerve autografts by Yusuke Katayama; Rivelino Montenegro; Thomas Freier; Rajiv Midha; Jason S. Belkas; Molly S. Shoichet (505-518).
Despite spontaneous sprouting of peripheral axons after transection injury, peripheral regeneration is incomplete and limited to short gaps, even with the use of autograft tissue, which is considered to be the “gold” standard. In an attempt to obviate some of the problems associated with autografts, including limited donor tissue and donor site morbidity, we aimed to synthesize a synthetic nerve guidance channel that would perform as well as the nerve autograft. Given that the patency of the nerve guidance channel is critical for repair, we investigated a series of nerve guidance channel designs where patency and the resulting regenerative capacity were compared in a transected rat sciatic nerve injury model. Three tube designs were compared to autograft tissue: plain, corrugated and coil-reinforced tubes of poly(2-hydroxyethyl methacrylate-co-methyl methacrylate). Of the three designs, the coil-reinforced tubes demonstrated superior performance in terms of patency. By electrophysiology and histomorphometry, the coil-reinforced tubes demonstrated outcomes that were comparable to autografts after both 8 and 16 weeks of implantation. The nerve action potential (NAP) velocity and muscle action potential (MAP) velocity for the coil-reinforced PHEMA-MMA tube was 54.6±10.1 and 10.9±1.3 m/s, respectively at 16 weeks, which was statistically equivalent to those of the autograft at 37.5±7.9 and 11.3±2.0 m/s. The axon density in the coil-reinforced tube was 2.16±0.61×104  axons/mm2, which was statistically similar to that of the autograft of 2.41±0.62×104  axons/mm2 at 16 weeks. These coil-reinforced tubes demonstrated equivalence to autografts for nerve regeneration, demonstrating the importance of channel design to regenerative capacity and more specifically the impact of patency to regeneration.
Keywords: Peripheral nerve regeneration; PHEMA; Patency; Tissue engineering;

We have previously shown that a novel synthetic hydrogel channel composed of poly(2-hydroxyethyl methacrylate-co-methyl methacrylate) (pHEMA-MMA) is biocompatible and supports axonal regeneration after spinal cord injury. Our goal was to improve the number and type of regenerated axons within the spinal cord through the addition of different matrices and growth factors incorporated within the lumen of the channel. After complete spinal cord transection at T8, pHEMA-MMA channels, having an elastic modulus of 263±13 kPa were implanted into adult Sprague Dawley rats. The channels were then filled with one of the following matrices: collagen, fibrin, Matrigel™, methylcellulose, or smaller pHEMA-MMA tubes placed within a larger pHEMA-MMA channel (called tubes within channels, TWC). We also supplemented selected matrices (collagen and fibrin) with neurotrophic factors, fibroblast growth factor-1 (FGF-1) and neurotrophin-3 (NT-3). After channel implantation, fibrin glue was applied to the cord-channel interface, and a duraplasty was performed with an expanded polytetrafluoroethylene (ePTFE®) membrane. Controls included animals that had either complete spinal cord transection and implantation of unfilled pHEMA-MMA channels or complete spinal cord transection. Regeneration was assessed by retrograde axonal tracing with Fluoro-Gold, and immunohistochemistry with NF-200 (for total axon counts) and calcitonin gene related peptide (CGRP, for sensory axon counts) after 8 weeks survival. Fibrin, Matrigel™, methylcellulose, collagen with FGF-1, collagen with NT-3, fibrin with FGF-1, and fibrin with NT-3 increased the total axon density within the channel (ANOVA, p < 0.05 ) compared to unfilled channel controls. Only fibrin with FGF-1 decreased the sensory axon density compared to unfilled channel controls (ANOVA, p < 0.05 ). Fibrin promoted the greatest axonal regeneration from reticular neurons, and methylcellulose promoted the greatest regeneration from vestibular and red nucleus neurons. With Matrigel™, there was no axonal regeneration from brainstem motor neurons. The addition of FGF-1 increased the axonal regeneration of vestibular neurons, and the addition of NT-3 decreased the total number of axons regenerating from brainstem neurons. The fibrin and TWC showed a consistent improvement in locomotor function at both 7 and 8 weeks. Thus, the present study shows that the presence and type of matrix contained within synthetic hydrogel guidance channels affects the quantity and origin of axons that regenerate after complete spinal cord transection, and can improve functional recovery. Determining the optimum matrices and growth factors for insertion into these guidance channels will improve regeneration of the injured spinal cord.
Keywords: Axonal regeneration; Methylcellulose; Matrigel™; Fibroblast growth factor-1; Neurotrophin-3; Functional recovery;