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Regeneration of Sciatic Nerve Across 15mm Gap by Use of a Polymeric Template
Abstract
A porous biodegradable collagen-glycosaminoglycan bridge induced regeneration of myelinated and unmyelinated axons over large distances (15 mm) between the severed ends of the adult rat sciatic nerve. The newly formed strand of nerve tissue was well vascularized and sheathed in connective tissue 6 weeks following implantation of the polymeric guide. Nerve tissue did not grow in the absence of the polymeric guide.
... Recent advancements in tissue engineering have led to the development of various techniques for fabricating collagen scaffolds. Freezedrying processing was pioneered by Yannas et al. to create porous collagen-glycosaminoglycan scaffolds, which could be packed with therapeutically active chemicals that are released as the scaffold degrades [97]. Other customized features of scaffolds include thermal-responsiveness, pH-sensitivity, and controlled drug release in injury sites [98,99]. ...
Neural tissue engineering (NTE) has made remarkable strides in recent years and holds great promise for treating several devastating neurological disorders. Selecting optimal scaffolding material is crucial for NET design strategies that enable neural and non-neural cell differentiation and axonal growth. Collagen is extensively employed in NTE applications due to the inherent resistance of the nervous system against regeneration, functionalized with neurotrophic factors, antagonists of neural growth inhibitors, and other neural growth-promoting agents. Recent advancements in integrating collagen with manufacturing strategies, such as scaffolding, electrospinning, and 3D bioprinting, provide localized trophic support, guide cell alignment, and protect neural cells from immune activity. This review categorises and analyses collagen-based processing techniques investigated for neural-specific applications, highlighting their strengths and weaknesses in repair, regeneration, and recovery. We also evaluate the potential prospects and challenges of using collagen-based biomaterials in NTE. Overall, this review offers a comprehensive and systematic framework for the rational evaluation and applications of collagen in NTE.
... If the electrodes are implanted into nerves, their appropriately designed surfaces should incite the neurons to adhere to the electrodes and to regenerate their axons along the electrodes. First investigations for the purpose of peripheral nerve regeneration were performed with biodegradable polymers derived, e.g. from extracts of the extracellular matrix (Yannas et al., 1987; Aebischer, 1988; Chang et al., 1989). For a rational design of regeneration-promoting surfaces, it is necessary to ®nd out the key structures which give rise to the desired behaviour of cells, e.g. ...
Neuronal cells are unique within the organism. In addition to forming long-distance connections with other nerve cells and non-neuronal targets, they lose the ability to regenerate their neurites and to divide during maturation. Consequently, external violations like trauma or disease frequently lead to their disappearance and replacement by non-neuronal, and thus not properly functioning cells. The advent of microtechnology and construction of artificial implants prompted to create particular devices for specialised regions of the nervous system, in order to compensate for the loss of function. The scope of the present work is to review the current devices in connection with their applicability and functional perspectives. (1) Successful implants like the cochlea implant and peripherally implantable stimulators are discussed. (2) Less developed and not yet applicable devices like retinal or cortical implants are introduced, with particular emphasis given to the reasons for their failure to replace very complex functions like vision. (3) Material research is presented both from the technological aspect and from their biocompatibility as prerequisite of any implantation. (4) Finally, basic studies are presented, which deal with methods of shaping the implants, procedures of testing biocompatibility and modification of improving the interfaces between a technical device and the biological environment. The review ends by pointing to future perspectives in neuroimplantation and restoration of interrupted neuronal pathways.
... Presently, we are examining methods to maintain even dispersion of filaments within the guidance chamber. Numerous investigators have studied regeneration across extended gap lesions (longer than 1.4 cm) in the rat model (Williams et al., 1987; Yannas et al., 1987; Madison et al., 1988; Kakinoki et al., 1997 Kakinoki et al., , 1998 Hadlock et al., 1998; Arai et al., 2000). To our knowledge, none had reported regeneration across these gap distances with empty tube controls. ...
After injury, axonal regeneration occurs across short gaps in the peripheral nervous system, but regeneration across larger gaps remains a challenge. To improve regeneration across extended nerve defects, we have fabricated novel microfilaments with the capability for drug release to support cellular migration and guide axonal growth across a lesion. In this study, we examine the nerve repair parameters of non-loaded filaments. To examine the influence of packing density on nerve repair, wet-spun poly(L-Lactide) (PLLA) microfilaments were bundled at densities of 3.75, 7.5, 15, and 30% to bridge a 1.0-cm gap lesion in the rat sciatic nerve. After 10 weeks, nerve cable formation increased significantly in the filament bundled groups when compared to empty-tube controls. At lower packing densities, the number of myelinated axons was more than twice that of controls or the highest packing density. In a consecutive experiment, PLLA bundles with lower filament-packing density were examined for nerve repair across 1.4- and 1.8-cm gaps. After 10 weeks, the number of successful regenerated nerves receiving filaments was more than twice that of controls. In addition, nerve cable areas for control groups were significantly less than those observed for filament groups. Axonal growth across 1.4- and 1.8-cm gaps was more consistent for the filament groups than for controls. These initial results demonstrate that PLLA microfilaments enhance nerve repair and regeneration across large nerve defects, even in the absence of drug release. Ongoing studies are examining nerve regeneration using microfilaments designed to release neurotrophins or cyclic AMP.
... Unexpectedly, the ARTICLE IN PRESSTable 1 Regenerative activity (incremental axon outgrowth across nerve chamber) of several tubulated configurations compared to silicone tube standard a Experimental variable X ; that affects outgrowth of axons across a gap L c in presence of X (mm) L c in absence of X (mm) Shift length, DL (mm) b References A. Effects of use of nerve chamber, insertion of distal stump and ligation of distal tube end Collagen tube vs. no tubulation X13.4 p6.0 >7.4 [33] Distal stump inserted vs. open-ended tube 11.7 p6.0 >57 [16] Distal stump inserted vs. ligated distal end 11.7 p6.0 >57 [16] B. Tube wall composition Silicone tube (standard) 9.7 (9.7) c 0 EVA copolymer d vs. silicone standard p11.0 (9.7) c p1.3 [35] PLA, plasticized e vs. silicone standard X13.4 (9.7) c X3.7 [18] LA/e-CPL f vs. silicone standard X13.4 (9.7) c X3.7 [25] Collagen tube vs. silicone tube X13.4 8.0 X5.4 [27] Collagen tube degrading optimally vs. silicone tube est. 25.5 (9.7) c est. >15 estimated from [36] C. Tube wall permeability Cell-permeability vs. impermeability X19.4 13.4 X6.0 [37] Cell-permeability vs. protein-permeability X11.4 13.4 X3.9 [38] Protein-permeability vs. impermeability 7.5 8.9 À1.4 [38,39] D. Schwann cell suspensions Schwann cell suspension vs. PBS 21.4 p14.0 X7.4 [40] E. Tube filling: solutions of proteins bFGF vs. no factor 14.3 p11.0 >3.3 [35] NGF vs. cyt C g 11.1 10.4 0.7 [41] aFGF vs. no factor 15.5 p11.0 X4.5 [42] F. Tube filling: gels based on ECM components Fibronectin vs. cyt C g 19.1 18.6 0.5 [24] Laminin vs. cyt C g 18.6 18.6 0 [24] G. Tube filling: insoluble substrates Collagen-GAG matrix vs. no matrix 16.1 p11.0 X5.1434445 Oriented fibrin matrix across gap vs. oriented matrix only adjacent to each stump 15.5 p11. 0 X4.5 [17] Early forming vs. late-forming fibrin matrix 15.5 12.5 3.0 [17] Axially vs. randomly oriented fibrin 11.4 p6.7 X4.7 [21] Rapidly degrading CG matrix (NRT h ) vs. no CG matrix X13.4 8.5 X4.9 [23,46,47] Axial vs. radial orientation of pore channels in NRT h X13.4 10.0 X3.4 [23,47] Laminin-coated collagen sponge vs. no laminin coating 11.7 p6.0 >5.7 [48] Polyamide filaments i vs. no filaments 18.4 p11.0 X7.4 [49] NRT h in collagen tube vs. silicone tube j >25 7.7 >17.3 [10] a Data from rat sciatic nerve. ...
Peripheral nerve regeneration has been studied in a variety of animal models. Of these, the nerve chamber model has clearly dominated. It has been used to generate a large base of data that, however, cannot be analyzed usefully due to lack of standardization of experimental conditions and assays. Lack of standardization of critical experimental parameters of the model has, however, greatly limited the opportunity to compare directly data from independent investigators; as a result, progress in understanding conditions for optimal nerve regeneration has been stunted. In this article, we provide an overview of the major experimental parameters that must be controlled in order to generate data from independent investigators that can be compared directly (normalized data). Such parameters include the gap length, animal species, and the identity of assays used to evaluate the product of the regenerative process. Use of the recently introduced concept of critical axon elongation, the gap length at which the probability of axonal outgrowth (reinnervation) across the gap is 50%, leads to generation of a normalized database that includes data from several independent investigators. Conclusions are drawn about the relative efficacy of the various biomaterials and devices employed. Nerve chamber configurations that had the highest regenerative activity were those in which the tube wall comprised collagen and certain synthetic biodegradable polymers rather than silicone, and was cell-permeable rather than protein-permeable. In addition, the following tube fillings showed very high regenerative activity: suspensions of Schwann cells; a solution either of acidic or basic fibroblast growth factor; insoluble ECM substrates rather than solutions or gels; polyamide filaments oriented along the tube axis; highly porous, insoluble analogs of the ECM with specific structure and controlled degradation rate.
In response to severe injury to the skin or peripheral nerves, adult mammals typically undergo an irreversible repair process that results in contraction and the formation of non-physiologic scar tissue. However, recent advancements with induced regeneration using biologically active scaffolds have demonstrated that it is possible to intervene during the healing process to partially or near-completely restore the physiologic function of damaged skin or peripheral nerves. The aim of these scaffolds is to promote regeneration and minimize the contraction and scar formation mediated by stromal fibroblasts. For instance, some scaffolds appear to downregulate TGF-β signaling, a key inductor of myofibroblasts which promote contraction and scar formation. Two collagen-based and three synthetic-based regenerative devices have been approved by the Food and Drug Administration (FDA), two for the regeneration of the skin and three for the regeneration of peripheral nerves. Increasingly, these devices are establishing themselves as a viable alternative to autografting.
Meniscus replacement remains a difficult prospect. Attempts to use artificial materials have led to synovitis and articular cartilage wear. Autografts generally have failed to protect the tibial surface and have led to scar tissue formation. Allografts of meniscus cartilage from cadavers generally have shrunk in animal and human tests to date, have failed in arthritic knees, and may be complicated by immunologic reaction. Pain relief has been reported in pristine knees, however. Collagen scaffolds for meniscus regeneration are showing promise in current human clinical trials.
Injury to the mammalian fetus is reversible during early stages of gestation and the spontaneous wound response is capable of restoring the structure and function of the original organ, a process called regeneration . By contrast, the unimpaired response to severe injury in adult mammals is an irreversible repair process leading to closure of the injured site by contraction and formation of scar, a nonphysiological tissue. The consequences of irreversible healing at the organ scale are far-reaching: they typically result in an essentially nonfunctional organ. Numerous approaches have been investigated to restore the loss of organ function in adults following irreversible injury. These strategies include transplantation, autografting, implantation of permanent prostheses, the use of stem cells, in vitro synthesis of the organ, and regenerative medicine (Yannas, Tissue and organ regeneration in adults. Springer; 2001). The last of these strategies is also referred to as induced organ regeneration , or the recovery of physiological structure and function of nonregenerative tissues in an organ (also known as de novo synthesis) by use of elementary reactants, such as biologically active scaffolds, either unseeded or seeded with cells. There is accumulating evidence that the spontaneous healing process of an injured organ in the adult mammal can be modified to yield a partially or completely regenerated organ. Regenerative medicine is an emerging field of study involving the implantation of biomaterials to facilitate formation (regeneration) of tissue in vivo. This field is undergoing rapid growth at this time, as evidenced by observation of regeneration or reported progress in on-going research efforts in a wide range of organs including skin (Butler and Orgill, Adv Biochem Eng Biotechnol 94:23-41, 2005), conjunctiva (Hatton and Rubin, Adv Biochem Eng Biotechnol 94:125-140, 2005), peripheral nerves (Zhang and Yannas, Adv Biochem Eng Biotechnol 94:67-89, 2005), bone (Mistry and Mikos, Adv Biochem Eng Biotechnol 94: 1-22, 2005), heart valves (Rabkin-Aikawa et al., Adv Biochem Eng Biotechnol 94:141-178, 2005), liver (Takimoto et al., Cell Transplant 12(4):413-421, 2003) articular cartilage (Kinner et al., Adv Biochem Eng Biotechnol 94:91-123, 2005), urological organs (Atala, Adv Biochem Eng Biotechnol 94:179-208, 2005), and the spinal cord (Verma and Fawcett, Adv Biochem Eng Biotechnol. 94:43-66, 2005). © Springer Science+Business Media, LLC 2012. All rights reserved.
A continuing study of preferences of elongating axons and Schwann cells for specific matrix features has revealed an apparently critical structural requirement. Well-defined, chemical analogs of ECM based on a collagen-glycosaminoglycan (CG) copolymer were used to bridge a 10-mm gap between cut ends of the rat sciatic nerve. The nerve stumps and the CG matrix bridging them were ensheathed in a silicone tube. Electrophysiological properties of regenerating motor nerve fibers innervating the plantar flexor muscles were serially monitored over 40 weeks following surgery. The results suggest that functional recovery of motor function requires the presence of an average pore diameter of order 1 µm. These results pose novel questions about the nature of cell-matrix interactions during nerve regeneration.
Tissue-engineering materials and approaches have recently been proposed as the future direction for tissue and organ regeneration. Ideal surgical reconstructions would replace biological tissues like autogenous tissues. In many cases autogenous reconstructions are limited by the nonoptimal availability of usable autogenous tissues or by donor site morbidity. Allografts are susceptible to increasing risks of infection and to transmission by infectious diseases or agents. Alloplastic materials (synthetic implants) offer no potential for direct infectious agents transmission but increase the susceptibility to indirect infections and inadequate long-term immunological interactions with the host and in some cases are plagued with clear failures, rejections, and uncertain long-term functioning. As a consequence, alternative materials and therapies have been investigated with the hope of benefiting from the advantages of living biological tissues or from those of artificial materials and structures.
We review the details of preparation and of the recently elucidated mechanism of biological (regenerative) activity of a collagen scaffold (dermis regeneration template, DRT) that has induced regeneration of skin and peripheral nerves (PN) in a variety of animal models and in the clinic. DRT is a 3D protein network with optimized pore size in the range 20–125 µm, degradation half-life 14 ± 7 d and ligand densities that exceed 200 µM α1β1 or α2β1 ligands. The pore has been optimized to allow migration of contractile cells (myofibroblasts, MFB) into the scaffold and to provide sufficient specific surface for cell–scaffold interaction; the degradation half-life provides the required time window for satisfactory binding interaction of MFB with the scaffold surface; and the ligand density supplies the appropriate ligands for specific binding of MFB on the scaffold surface. A dramatic change in MFB phenotype takes place following MFB-scaffold binding which has been shown to result in blocking of wound contraction. In both skin wounds and PN wounds the evidence has shown clearly that contraction blocking by DRT is followed by induction of regeneration of nearly perfect organs. The biologically active structure of DRT is required for contraction blocking; well-matched collagen scaffold controls of DRT, with structures that varied from that of DRT, have failed to induce regeneration. Careful processing of collagen scaffolds is required for adequate biological activity of the scaffold surface. The newly understood mechanism provides a relatively complete paradigm of regenerative medicine that can be used to prepare scaffolds that may induce regeneration of other organs in future studies.
There is a diverse range of materials and methods available for the immobilization of biomolecules and cells on or within biomaterial supports. This chapter explains various classes of materials used in medicine. Metallic implant materials have a significant economic and clinical impact on the biomaterials field. Apart from orthopedics, there are other markets for metallic implants and devices including (1) oral and maxillofacial surgery, for example, dental implants, craniofacial plates, and screws and (2) cardiovascular surgery, for example, the parts of artificial hearts, pacemakers, balloon catheters, valve replacements, and aneurysm clips. The chapter also introduces the concepts of polymer characterization and property testing as they are applied to the selection of biomaterials. It provides a table that compares some of the biomolecule immobilization techniques—physical and electrostatic adsorption, cross linking, entrapment, and covalent binding.
We have investigated the repair of peripheral nerves in animal models using tubular guiding conduits. The materials used to fabricate the nerve conduits and their physicochemical and mechanical characteristics can influence the extent, rate and morphology of regeneration. Permeability of the conduit membranes is one parameter which seems to play an important role in nerve regeneration. In the present study, two types of nerve conduits were developed from bovine tendon collagen with distinctly different permeabilities. The permeability of the conduit membranes was determined by diffusion of various sized molecules across these membranes. One type of conduit had pores which only allowed small molecules such as glucose to pass (small pore collagen conduits). The other type had pores which were readily permeable to macromolecules such as bovine serum albumin (large pore collagen conduits). The large pore collagen conduits supported nerve regeneration to a greater degree than the small pore collagen conduits when tested in mice to bridge 4 mm gaps of the sciatic nerve. Studies in rats and primates suggested that large pore collagen conduits worked as effectively as nerve autografts in terms of physiological recovery of motor and sensory responses. The results of in vitro and in vivo studies of these conduits represent a significant step towards our specific aim of developing suitable off-the-shelf prostheses for clinical repair of damaged peripheral nerves.
Tendon is a specialized dense connective tissue that links muscle to bone and allows for the transmission of muscle contraction forces to the bone for skeletal locomotion; for example, the Achilles tendon, one of the largest tendons in the body, links the triceps surae muscle, the grouping of the gastrocnemius, the soleus, and the plantaris muscles to the calcaneous bone. There is a compelling need for engineered tendon. This is because any defect large enough to preclude a direct suture repair of apposed tendon ends results in a reparative tissue with inadequate mechanical properties. Promising results have been obtained in preliminary studies of tenocyte and mesenchymal stem cell–seeded matrices. Moreover, there appear to be animal models in which the use of implants to facilitate tendon regeneration can be critically evaluated. Several strategies for engineering tendon based on promising findings in vitro and in animal models are currently being pursued. The fact that no treatment modality in human subjects or animal models has yet yielded a reparative tissue with the histological characteristics specific to tendon indicates the magnitude of the challenge to be met.
Results of implantation studies in a variety of animal tissue models demonstrate that the rate of biogradation of a collagen scaffold should parallel the rate of wound healing observed in particular anatomic sites. This rapid degradation maximizes tissue regeneration and minimizes encapsulation of the implant. The following paper reviews the effects of crosslinking on the rate of tissue ingrowth and regeneration. In addition, preliminary mechanical data on newly developed soluble type I collagen fibers is presented as a possible advance in the production of high strength collagen based tissue scaffolds.
Current strategies of regenerative medicine are focused on the restoration of pathologically altered tissue architectures by transplantation of cells in combination with supportive scaffolds and biomolecules. In recent years, considerable interest has been given to biologically active scaffolds which are based on similar analogs of the extracellular matrix that have induced synthesis of tissues and organs. To restore function or regenerate tissue, a scaffold is necessary that will act as a temporary matrix for cell proliferation and extracellular matrix deposition, with subsequent ingrowth until the tissues are totally restored or regenerated. Scaffolds have been used for tissue engineering such as bone, cartilage, ligament, skin, vascular tissues, neural tissues, and skeletal muscle and as vehicle for the controlled delivery of drugs, proteins, and DNA. Various technologies come together to construct porous scaffolds to regenerate the tissues/organs and also for controlled and targeted release of bioactive agents in tissue engineering applications. In this paper, an overview of the different types of scaffolds with their material properties is discussed. The fabrication technologies for tissue engineering scaffolds, including the basic and conventional techniques to the more recent ones, are tabulated.
Acute or chronic injury to an organ is followed by a spontaneous healing process. Injury to the mammalian fetus is reversible
during early stages of gestation; the spontaneous wound response is capable of restoring the structure and function of the
original organ (regeneration). In contrast, the unimpaired response of adults to severe injury is an irreversible process
leading to closure of the injured site by contraction and formation of scar, a nonphysiological tissue (repair). The consequences
of irreversible healing at the organ scale are far reaching: they often result in an essentially nonfunctional organ.
Die Entwicklung von Tieren beginnt mit einer einzigen Zelle, die sich teilt; die neuen Zellen differenzieren zu hochspezifischen Geweben, Organen und Gliedern, und der kleine, aber funktionstüchtige Organismus erreicht irgendwann seine volle Größe. Während dieser Entwicklung durchlaufen auch die extrazellulären Matrices (ECM), d. h. komplexe makromolekulare Netzwerke, drastische Veränderungen. Matrixtransformationen steuern mitunter die viel besser untersuchten Änderungen von Anzahl und Typus der differenzierenden Zellen. ECM-Netzwerke werden meist enzymatisch zu Oligopeptiden abgebaut und danach neu synthetisiert (umgestaltet), wobei sich unlösliche und nicht diffundierende makromolekulare Strukturen bilden, die vielzelligen Systemen ihre Form verleihen. Reife ECM wie Haut, Sehnen, Knorpel und Blutgefäße geben Organen und Geweben Steifheit und Stabilität. Eine Umgestaltung der ECM tritt während der Wundheilung auch in erwachsenen Organismen auf. Der Einsatz von synthetischen ECM-Analoga kann helfen, die Bedeutung der ECM für die Entwicklung oder Wundheilung zu verstehen. Es wurden einfache chemische Analoga synthetisiert, von denen einige eine bemerkenswerte biologische Aktivität aufweisen, darunter eins, das die partielle Regeneration der Haut sowohl bei erwachsenen Meerschweinchen als auch beim Menschen induziert. Mit einem ähnlichen ECM-Analogon wurden auch periphere Nerven regeneriert. Derartige verletzte Hautpartien und Nerven regenerieren sich bei Wirbeltieren nicht spontan. Verfahren zur Herstellung der ECM-Analoga mit maßgeschneiderten physikalisch-chemischen Eigenschaften wie Geschwindigkeit des enzymatischen Abbaus nach Implantation, Porenstruktur und Grad der Collagenkristallinität werden in diesem Aufsatz zusammengefaßt. ECM-Analoga zeigen völlig neue Wege bei der Behandlung von ernsten Organfehlfunktionen und Organverlust. Ein ECM-Analogon ist bereits die Basis einer klinischen Behandlung von Patienten mit starken Brandverletzungen. Eine Interpretation der Ergebnisse führt zu einer Hypothese über die Natur der ECM während der Entwicklung. Da biologische Aktivität nur dann beobachtet wird, wenn die physikalisch-chemischen Parameter innerhalb enger Grenzen liegen, könnte man die ECM als einen unlöslichen Wachstumsfaktor beschreiben, der für die Synthese von Gewebe spezifisch ist. Möglicherweise benötigt jedes Gewebe zur Entwicklung eine eigene ECM.
Methods for engineering the regeneration of peripheral nerve in lesions have generally focused on the implementation of tubes as implants to bridge the defect. Previous study has shown that a highly porous analog of the extracellular matrix of a specific pore size range, ensheathed by a silicone tube, enhanced the regeneration of axons across gaps of 10 mm and greater in a transected adult rat sciatic nerve model. This study reports the histological findings resulting from implantation of a fully degradable collagen device comprising the collagen-glycosaminoglycan (GAG) analog in a collagen tube in a 10-mm gap in this animal model. Silicone tubes, with and without the collagen-GAG matrix, served as controls. Results indicated that axons had regrown into the midsection of the gap in all prostheses by 30 weeks; however, in the presence of the collagen-GAG matrix, the number and size of the axons appeared to increase. A layer of fibrous tissue approximately 100 μm thick, which contained fibroblasts, surrounded the silicone tubes but was not visible along the tube wall in any of the collagen tube prostheses. These findings show the promise of a fully degradable prosthesis for facilitating regeneration following peripheral nerve injuries.
In previous studies, we have examined the substrate preferences of peripheral nervous system (PNS) axons elongating within a 15-mm gap in the rat sciatic nerve. Gaps were bridged with tubes filled with one of several porous collagen-glycosaminoglycan (GAG) copolymers. In this study, we have attempted to determine if this method for PNS regeneration can be used to study regeneration of axons in the central nervous system (CNS). A 5-mm gap was created in the midthoracic spinal cord of adult rats. Animals were divided into groups in which the gap was bridged with a collagen tube filled with one of two experimental collagen-GAG copolymers or bridged with an unfilled collagen tube. Lesions that were not bridged by a tubular device were included as controls. Rats were sacrificed at 30 days, and spinal cords, including the lesion site, were removed and studied histologically. Large numbers of myelinated axons were observed regrowing within the gap in animals of all groups. The presence of collagen tubes modified the organization and orientation of the fibrous component of reparative tissue and the migration pattern of astrocytes in the wound site. The results suggest that entubulation of the transected spinal cord is a useful technique for studying the substrate preferences of regenerating CNS axons.
Animal development starts as a single cell which proliferates into several new cells; these differentiate into highly specialized tissues, organs, and limbs; and the small but functioning organism eventually grows into its full scale. Throughout development the extracellular matrices, which are complex macromolecular networks, also undergo dramatic changes. Matrix transformations occasionally control the much more well-studied changes in number and type of differentiating cells. Extracellular matrix (ECM) networks are typically broken down enzymatically to oligopeptides and are then resynthesized (remodeled) to form insoluble and nondiffusible macromolecular structures which confer stability of shape to multicellular systems. Mature ECM, such as skin, tendon, cartilage, and blood vessels, provides stiffness and strength to tissues and organs. Remodeling of ECM also occurs in adult organisms, during wound healing. An understanding of the role that ECM plays during development or wound healing can be obtained by use of synthetic ECM analogues. Several simple chemical ECM analogues have been synthesized and a few have been found to possess remarkable biological activity. One of these analogues has induced the partial regeneration of skin in an adult guinea pig wound model as well as in man. Peripheral nerve has been regenerated in another animal model by use of a similar ECM analogue. In all these mammalian lesions it is well-known that regeneration does not occur spontaneously. These analogues are graft copolymers of collagen and chondroitin 6-sulfate (a glycosaminoglycan) in the state of highly hydrated and covalently cross-linked gels. Procedures are summarized for synthesis of copolymers with adjusted physicochemical properties, such as the rate at which they degrade enzymatically when implanted, the elements of their pore structure, and the degree of collagen crystallinity. ECM analogues have provided a novel window into the complexities of morphogenesis and regeneration and they have pointed towards entirely new directions in the medical treatment of serious organ dysfunction and organ loss. An ECM analogue has already become the basis of a new clinical treatment for massively burned patients. An interpretation of the results leads to a hypothesis about the nature of ECM during development. Since biological activity appears only when the physicochemical parameters fall within very narrow limits, it is intriguing to speculate that these experiments describe a single insoluble growth factor which is specific for skin synthesis. Such an insoluble growth factor appears to be just as essential to skin development as are the much more well-known soluble growth factors. A different ECM analogue appears to induce nerve regeneration, possibly because each tissue requires its own developmentally active ECM.
Regenerative medicine aims at inducing the formation of physiological tissues, in cases where the spontaneous healing response following a severe injury or disease leads to wound closure via contraction and synthesis of scar or fibrotic tissue. The suppression of both wound contraction and scar synthesis, with the simultaneous synthesis of physiological tissues, might be achieved by implanting a porous macromolecular scaffold within the site of injury, able to host cells and guide their behavior toward regeneration. Tubular scaffolds, reconnecting the proximal and distal stumps of a transected peripheral nerve, demonstrated to be able to induce regeneration of the lost nerve trunk. Experimental evidence from independent investigations shows that protein-permeable porous tubes, of different types, perform better as regenerative templates when their pore size is also cell-permeable (>10 μm). Moreover, the regenerative potential of the porous conduit may be further enhanced when inserting a substrate with longitudinally oriented pores within it, since such an oriented construct provides physical support and guide to the growth of neural structures across the site of injury. This study focused on the production of collagen-based conduits and matrices with a micropatterned porosity, suitable for use as nerve regenerative templates. The manufacturing techniques for the production of tubular and cylindrical scaffolds, with controlled pore size and orientation, were based on a freeze-drying process. Tubular scaffolds possessed a radially oriented porosity with a radial gradient of pore sizes, ranging from cell-permeable pores at the inner tube wall to cell-impermeable pores at the outer one. Cylindrical scaffolds exhibited nearly axially oriented pores, with a pore size depending on the freezing rate as well as on the collagen concentration. Due to their peculiar porous structures, such scaffolds are expected to enhance the regenerative capacity of transected peripheral nerves as well as to lead to a better understanding of the cellular mechanisms underlying peripheral nerve regeneration.
In the United States more than 200 000 people per year are treated for severe peripheral nerve injuries that require surgical intervention. Functional recovery of motor and sensory capability is limited after autografting, the most common surgical intervention for severe peripheral nerve injuries. The process of peripheral nerve regeneration has been studied extensively in a variety of animal models using a tubular conduit. This model has been used to generate a large base of data from a wide variety of experimental devices; however, this data has not been analyzed comparatively due to a lack of standardization of experimental conditions, assays, and reported measures of the quality of regeneration. As a result, progress in understanding conditions for optimal nerve regeneration has been stunted and the optimal characteristics for such an implant have not been identified. So while tubulation repair of a transected peripheral nerve presents an attractive alternative to autograft, it has not yet shown the ability to satisfactorily restore lost function. In this article, we provide an overview of mammalian wound healing following severe injury, the physiology of the peripheral nervous system, the standardized wound models used to study peripheral nerve regeneration, and the critical axon elongation criteria and how it can be used to directly compare results from dissimilar studies. We complete this review article with a description of the critical features of tubular implants used to induce peripheral nerve regeneration that can be optimized in order to improve the quality of regeneration.
With increasing fetal development, the mechanism of mammalian wound healing transitions from regeneration to a repair process characterized by organized wound contraction and scar synthesis. Recently, a variety of tissue-engineering constructs have been developed to block the contraction and scar formation mechanisms of repair, and to induce regeneration following injury with similar mechanisms of action observed for both the skin and peripheral nervous system (PNS). Such constructs, mostly scaffolds that are analogs of extracellular matrix and possess specific biological activity, have become the basis of studies of in vivo synthesis of tissues and organs. In this article, we provide an overview of mammalian wound healing processes following severe injury as well as a description of the tissue triad and the regenerative capacity of the three distinct tissue types that comprise the triad. We also discuss the critical structural elements of an active extracellular matrix analog that induces regeneration, and describe the use of standardized wound models for study of in vivo regeneration processes. We conclude this review describing recent data from studies utilizing active extracellular matrix analogs (scaffolds) that have shown regenerative activity.
Skin wounds, particularly burns, affect a large population each year. The injury response to skin wounds depends both of the severity of the wound and the animal affected. The typical organismic response to an injury at the organ-scale is cell-mediated wound contraction and synthesis of nonphysiologic tissue (scar); this process is termed repair. Regeneration of lost or damaged tissue describes a process marked by synthesis of physiologic (normal, functional) tissue in the wound site. With increasing organism development, wound closure depends increasingly less on regeneration and correspondingly more on contraction and scar formation. A variety of tissue engineering constructs have been utilized to prevent contraction and scar formation and induce regeneration following skin wounds. Experimental experience has identified a number of specific criteria to maximize the bioactivity of such a graft, leading to maximal regeneration. These criteria as well as the morphology, functionality, and regenerative capacity of the three tissues that comprise skin will be detailed. In addition to the description of the organismic response to skin injuries and the current and historical clinical treatments for such injuries, five specific devices developed to treat severe skin injuries, including the design and manufacture of each device as well as the attendant experimental and clinical successes, will be detailed.
Use of collagenous substrates for growth of attachment-dependent and attachment-independent cells have been reported in the literature. Growth of fibroblasts and epithelial cells on collagen sponges and fibers has been previously studied in our laboratory. This article reports the result of studies of neurite outgrowth on etched collagen fibers in cell culture. Collagen fibers with a mean diameter of about 134 μm were etched on glass coverslips using a collagenase solution until individual fibrils of 1 μm in diameter were observed. The kinetics of etching were optimized at a calcium chloride concentration of 5 mM and a collagenase activity of 300 unit/mL. At room temperature an etching time of 10 h maximized the number of fibrils exposed per fiber. Cell culture studies on etched collagen fibers indicate that neurites from rat fetal cortical neurons elongate along the longitudinal axes of collagen fibrils. In some instances the cell body observed was not on top of the collagen fibrils but situated between two parallel fibrils. Results of these experiments indicate that growth of neurites along collagen fibrils in cell culture may be a useful model to evaluate neurite extension in the presence of neurotrophic factors as well as to study optimization of substrates for nerve regeneration.
The presence of contractile cells, their organization around regenerating nerve trunks, and the hypothetical effect of these organized structures on the extent of regeneration across a tubulated 10-mm gap in the rat sciatic nerve were investigated. Collagen and silicone tubes were implanted both empty and filled with a collagen-glycosaminoglycan (GAG) matrix. Nerves were retrieved at 6, 30, and 60 weeks postoperatively and time-dependent values of the nerve trunk diameter along the tubulated length were recorded. The presence of myofibroblasts was identified immunohistochemically using a monoclonal antibody to -smooth muscle actin. Myofibroblasts were circumferentially arranged around the perimeter of regenerated nerve trunks, forming a capsule which was about 10 times thicker in silicone tubes than in collagen tubes. The nerve trunk diameter that formed inside collagen tubes was twice as large as that inside silicone tubes. In contrast, the collagen-GAG matrix had a relatively small effect on capsule thickness or diameter of regenerate. It was hypothesized that the frequency of successful bridging by axons depends on the balance between two competitive forces: the axial forces generated by the outgrowth of axons and nonneuronal cells from the proximal stump and the constrictive, circumferential forces imposed by the contractile tissue capsule that promote closure of the wounded stumps and prevent axon elongation. Because the presence of the collagen-GAG matrix has enhanced greatly the recovery of normal function of regenerates in silicone tubes, it was hypothesized that it accelerated axonal elongation sufficiently before the hypothetical forces constricting the nerve trunk in silicone tubes became sufficiently large. The combined data suggest a new mechanism for peripheral nerve regeneration along a tubulated gap. J. Comp. Neurol. 417:415–430, 2000. © 2000 Wiley-Liss, Inc.
In mammals, the skin normally responds to injury by the processes of wound contraction and scar formation. However, if a unique acellular dermal regeneration template is placed into the wound, wound contraction is strongly inhibited, and the skin undergoes a regenerative-type healing. It is proposed that these phenomena result from the disruption of a “mechanically coherent” organization of myofibroblasts within the wound. Evidence suggests that the dermal regeneration template prevents the formation of cell-extracellular matrix interactions (fibronexi), which foster the assemblage of a mechanically coherent wound bed. Additional studies support a similar mechanism for the enhanced regenerative-like response to peripheral nerves when extracellular matrix tubes are used to facilitate healing.
The nerve chamber model has dominated the experimental study of peripheral nerve (PN) regeneration with animal models as well as in several clinical applications, such as the treatment of paralysis of limbs following severe trauma. The two stumps resulting from nerve transection are inserted inside a tubular chamber made from one of several materials, occasionally filled with various substances, and the quality of the reconnected nerve is assayed. Recent use of methods for data reduction has led to generation of a large normalized database from independent investigations. Methods for data normalization (reduction) are based on systematic use of the critical axon elongation, Lc, the gap length between the transected stumps at which the frequency of reconnection is just 50% for a given configuration. Four theories are compared for their ability to explain the normalized data. Although the neurotrophic and contact guidance theories explain some of the data, combined use of the more recent microtube theory and pressure cuff theory appears capable of explaining a much larger data set. PN regeneration appears to be upregulated by chamber configurations that facilitate formation of basement membrane microtubes about 10–20 m in diameter, comprising linear columns of Schwann cells surrounded by basement membrane, into which axons elongate and eventually become myelinated. Regeneration is downregulated by experimental configurations that permit formation of a contractile cell (myofibroblast) capsule around the regenerating nerve that appears to restrict growth of a nerve trunk by application of circumferential mechanical forces. These two processes work competitively to regulate nerve regeneration in the chamber model.
There is currently no method to restore normal function in tendon injuries that result in a gap. The objective of this study was to evaluate the early healing of tendon defects implanted with a porous collagen–glycosaminoglycan (CG) matrix, previously shown to facilitate the regeneration of dermis and peripheral nerve. A novel animal model that isolates the tendon defect site from surrounding tissue during healing was employed. This model used a silicone tube to entubulate the surgically produced tendon gap of 10 mm, allowing for the evaluation of the effects of the analog of extracellular matrix on healing of tendon, absent the influences of the external environment. The results showed that tendon stumps induced synthesis of a tissue cable inside the silicone tube in both the presence and absence of CG matrix. The presence of the CG matrix, however, altered the process of tendon healing. Tubes filled with CG matrix contained a significantly greater volume of tissue at the time periods of evaluation: 3, 6, and 12 weeks. Granulation tissue persisted for a longer period of time in the lesion site of CG-filled defects, and the amount of dense fibrous tissue increased continuously during the period of study in defects filled with CG matrix. In contrast, the amount of dense fibrous tissue decreased after 6 weeks in originally empty tubes. In tubes that did not contain the CG matrix, the new tissue consisted of dense aggregates of crimped fibers with a wavelength and fiber bundle thickness that were significantly shorter than those in normal tendon, and consistent with the type of scar that is the end result of repair of many connective tissues. Although, CG-filled tubes contained dense fibrous tissue by 12 weeks, the tissue had no crimp. The CG matrix may have prolonged the synthesis of granulation tissue and delayed or prevented the formation of scar. Peer Reviewed http://deepblue.lib.umich.edu/bitstream/2027.42/63265/1/ten.1997.3.187.pdf
Peripheral neural regeneration, over a 10-mm transectional gap, was determined in 70 rabbit buccal divisions of the facial nerve using two entubational systems (semipermeable and impermeable silicone chambers) prefilled with three natural occurring media (serum, blood, and saline) during a 5-week period. The number of myelinated axonal regenerates at the midchamber and at 2 mm in the distal transected neural stump were counted in each group and compared to pooled myelinated axonal counts in 9 normal rabbit buccal divisions of the facial nerve. Semipermeable porous chambers had an overall greater regeneration success rate (75% vs. 42.8%) and regained, on the average, a higher number of myelinated axons (51.4% vs. 26.1%) than silicone chamber regenerates. Semipermeable chambers prefilled with serum or blood had significantly higher regeneration success rates, myelinated axonal counts, and percentages of neural innervation of the distal transected neural stump. Both entubational systems produced similar axonal counts with intraluminal saline. The highest overall success rate (93.7%) and average number of myelinated axons per chamber (3072) were achieved in semipermeable chambers prefilled with serum. The greatest variability in myelinated axonal counts (0 to 3266 axons) and percentage of distal stump innervation (5.5% to 98.1%) was seen in silicone chambers filled with saline. The percentage of myelinated axons from the midchamber that innervated the distal stump was greater in semipermeable chambers with blood (73%) and serum (54%) than in silicone saline chambers (43%). On the average, the distal stumps from semipermeable chambers filled with serum (47%) and blood (33.5%) regained a higher percentage of normal myelinated axonal counts than silicone-saline chambers (12.5%). These results suggest that both the construction of entubational chamber and the intraluminal medium can have significant influence on neurite regeneration. Semipermeable chambers prefilled with serum have a strong neurite-promoting potential in peripheral neural regeneration of rabbit facial nerves.
We sought to create a regeneration template for the meniscal cartilage of the knee to induce complete men iscal regeneration, and to develop the technique for implanting the prosthetic appliance in vivo. We de signed a resorbable collagen-based scaffold and con ducted in vitro and in vivo studies. In vivo, the scaffold was implanted in the knees of immature swine and mature canines and evaluated clinically, histologically, and biochemically. Because the canine stifle joint me niscus is more clinically relevant to the human menis cus, this paper emphasizes those results.
We studied 24 mixed breed dogs (14 males and 10 females) with an average weight of 25.5 kg (range, 20 to 35) that were obtained from a USDA-licensed sup plier. The dogs were deemed clinically and radiograph ically skeletally mature. None of the dogs had a preex isting knee joint abnormality. All dogs underwent an 80% subtotal resection of the medial meniscus bilater ally. A collagen template was implanted in one stifle (N = 24). The contralateral side served as a control: 12 dogs had a total resection alone and the other 12 dogs had an immediate replantation of the autologous meniscus.
Results were tabulated at 3, 6, 9, and 12 months. At final evaluation, before the animals were euthanized, the results were submitted for statistical analysis as well as histologic and biochemical analyses. The results demonstrated that a copolymeric collagen-based scaf fold can be constructed that is compatible with meniscal fibrochondrocyte growth in vitro and in vivo, that does not inhibit meniscal regeneration in an immature pig, and that may induce regeneration of the meniscus in the mature dog.
Although additional studies are necessary to perfect the scaffold and to evaluate the implant in the environ ment of the knee, these studies suggest that effective meniscal regeneration can be supported by an im planted collagen-based scaffold designed to support cellular ingrowth.
Biomaterials have made a great impact on medicine. However, numerous challenges remain. This paper discusses three representative areas involving important medical problems. First, drug delivery systems; major considerations include drug-polymer interactions, drug transformation, diffusion properties of drugs and, if degradation occurs, of polymer degradation products through polymer matrices developing a more complete understanding of matrix degradation in the case of erodible polymers and developing new engineered polymers designed for specific purposes such as vaccination or pulsatile release. Second, cell-polymer interactions, including the fate of inert polymers, the use of polymers as templates for tissue regeneration and the study of polymers which aid cell transplantation. Third, orthopaedic biomaterials, including basic research in the behaviour of chondrocytes, osteocytes and connective tissue-free interfaces and applied research involving computer-aided design of biomaterials and the creation of orthopaedic biomaterials.
The loss of tissue mass in humans has been conventionally treated as an irreversible change. Treatments have emphasized replacement of the missing function by use of a transplant, an autograft, tissue synthesized in vitro or, most commonly, by use of engineering devices based on biomaterials. During the last few years solid progress has been made in the area of tissue and organ regeneration. This new approach is based on the discovery that certain simple chemical analogs of extracellular matrices synthesized by graft copolymerization of a glycosaminoglycan onto type I collagen can induce synthesis of physiologic tissue in lesions which otherwise heal spontaneously by synthesis of scar tissue. This approach offers serious potential advantages over the alternatives listed above since the graft "grows out" of host tissue. However, regeneration in the adult mammal has been successfully demonstrated so far only in skin (human, guinea pig), sciatic nerve (rat) and the knee meniscus (dog).
Biologically active analogues of the extracellular matrix (ECM) are synthesized by grafting glycosaminoglycan (GAG) chains onto type I collagen, and by controlling the physicochemical properties of the resulting graft copolymer. Collagen-GAG ECM analogues have previously been shown to induce regeneration of the dermis in humans and the guinea pig, and of the rat sciatic nerve. Current studies have emphasized elucidation of the molecular mechanism through which tissue-specific ECM analogues induce regeneration. The contribution of the GAGs to the biological activity of the skin regeneration template was confirmed by studying the contribution of several GAGs to the inhibition of wound contraction in guinea pigs. The interaction between cells and the porous structure of an ECM analogue was studied with emphasis on the deformation of pores which occurs during wound contraction. The synthesis of scar, as well as of partly regenerated tissue which has a morphology between that appropriate for scar and for normal dermis, was quantitatively assayed for the first time using a laser light scattering technique. An ECM analogue which has been shown to be capable of inducing regeneration of functional sciatic nerve in the rat over a gap larger than 10 mm was incorporated in the design of a biodegradable implant for peripheral nerve regeneration.
Injuries to peripheral nerves innervating a limb cause paralysis, and can necessitate amputation. The inability of the nerves to regenerate spontaneously and the limitations of autograft procedures led to the development of treatments involving insertion of the nerve ends into prosthetic tubular devices. Previous work showed that 'entubulation' of the nerve ends in a silicone tube containing a specific porous, resorbable collagen-GAG (CG) copolymer, serving as an analog of extracellular matrix, improved regeneration compared to an empty silicone tube. However, long-term treatment with silicone tubes produced constriction that caused partial degradation of the regenerated axons; for this and other reasons, implementation of a nondegradable tube may require a second surgical procedure for removal. In this study the silicone tube was replaced with porous and non-porous collagen tubes in order to produce fully degradable devices. CG-filled collagen tubes and controls (CG-filled silicone tubes and empty collagen and silicone tubes) were implanted in a 10-mm gap in the rat sciatic nerve, with three rats in each group. The regeneration was evaluated after six weeks using light microscope images of cross sections of the nerve that were digitized and analyzed. Histograms of the diameters of the axons were generated and compared. The cellular response to the implanted biomaterials was assessed histologically, and immunohistochemistry was performed using an antibody to alpha-smooth muscle actin in order to determine the presence of myofibroblasts (contractile cells). Axonal regrowth was comparable in porous collagen, non-porous collagen, and silicone tubes filled with a CG matrix. These results support the implementation of a degradable collagen tube in place of a silicone device. Confirming earlier work, regeneration through the silicone and collagen tubes was enhanced by the CG copolymer, compared to empty tubes. A notable finding was a continuous layer of myofibroblasts on the surfaces of all of the six silicone tube prostheses, but on the inner surface of only one of six collagen tubes (Fisher's exact tests; P < 0.01). This is the first report of contractile capsules around silicone tubes, and supports the use of degradable collagen tubes in peripheral nerve regeneration. Macrophages were found bordering both the silicone and collagen tubes, and in the case of the collagen tubes, appeared to be participating in the regulation of the tubes.
Peripheral nerve regeneration was studied across a tubulated 10-mm gap in the rat sciatic nerve using histomorphometry and electrophysiological measurements of A-fiber, B-fiber, and C-fiber peaks of the evoked action potentials. Tubes fabricated from large-pore collagen (max. pore diameter, 22 nm), small-pore collagen (max. pore diameter, 4 nm), and silicone were implanted either saline-filled or filled with a highly porous, collagen-glycosaminoglycan (CG) matrix. The CG matrix was deliberately synthesized, based on a previous optimization study, to degrade with a half-life of about 6 weeks and to have a very high specific surface through a combination of high pore volume fraction (0.95) and relatively small average pore diameter (35 microm). Nerves regenerated through tubes fabricated from large-pore collagen and filled with the CG matrix had significantly more large-diameter axons, more total axons, and significantly higher A-fiber conduction velocities than any other tubulated group; and, although lower than normal, their histomorphometric and electrophysiological properties were statistically indistinguishable from those of the autograft control. Although the total number of myelinated axons in nerves regenerated by tubulation had reached a plateau by 30 weeks, the number of axons with diameter larger than 6 microm, which have been uniquely associated with the A-fiber peak of the action potential, continued to increase at substantial rates through the completion of the study (60 weeks). The kinetic data strongly suggest that a nerve trunk maturation process, not previously reported in studies of the tubulated 10-mm gap in the rat sciatic nerve, and consisting in increase of axonal tissue area with decrease in total tissue area, continues beyond 60 weeks after injury, resulting in a nerve trunk which increasingly approaches the structure of the normal control.
Simple chemical analogs of extracellular matrices have been synthesized by graft copolymerization of a glycosaminoglycan on to type I collagen. A few of these collagen-graft-glycosaminoglycan copolymers (CG copolymers) have diverted decisively the kinetics and mechanism of skin wound healing in animals and humans away from contraction and scar synthesis, towards the direction of skin regeneration. Detailed animal studies show that CG copolymers show maximum biological activity when the average pore diameter and the degradation rate in collagenase are controlled within critical limits. When seeded with a minimum number of cells these active copolymers induce regeneration of skin, including synthesis of a new epidermis and a new dermis in the correct anatomical relationship. Certain unseeded copolymers have also induced regeneration of peripheral nerve. Another copolymer has induced regeneration of the knee meniscus. The unusual biological activity of these copolymers has led to extensive, successful clinical testing of novel medical devices for the treatment of skin loss with severely burned patients.
The objective of this study was to investigate the presence of a contractile actin isoform, alpha-smooth muscle actin, in annulus fibrosus cells in situ and in two and three-dimensional cultures. Annulus fibrosus cells were isolated from healthy adult dogs, serial passaged, and then injected into porous collagen-glycosaminoglycan copolymers consisting of either type-I or type-II collagen. Alpha-smooth muscle actin was detected in the cells in tissue samples and in culture by immunohistochemistry. The number of cells and glycosaminoglycan content of the matrices were determined after 1, 7, and 14 days, and the diameters of the specimens were measured every 2 days. Although few annulus fibrosus cells in vivo displayed the presence of the alpha-smooth muscle actin isoform, most cells in two-dimensional culture demonstrated this phenotype. The contractile behavior of these cells was shown by the cell-mediated contraction of type-I collagen-glycosaminoglycan scaffolds after 8 days in culture. Glycosaminoglycan production was not significantly different in the seeded type-I matrices than in the unseeded matrices, whereas the seeded type-II matrices had a significant increase in glycosaminoglycan production between days 1 and 14 compared with the unseeded controls. This is the first report of both the expression of the contractile alpha-smooth muscle actin isoform in intervertebral disc cells and the ability of the cells to contract a collagen matrix. This finding could aid in better understanding the nature of cells in the annulus.
Ophthalmology has a long history of successful conventional biomaterial applications including viscoelastics, drug delivery vehicles, contact lenses, and a variety of implants. A myriad of further possibilities exists as the margins between conventional material concepts and natural tissues continue to blur, and biomaterials move closer to nature. Genetically engineered materials (for example, hyaluronic acid and fibrin tissue glues) harnessing the power and accuracy of biological systems in molecular synthesis are becoming commonplace. New synthetic surfaces capable of upregulating or downregulating biological responses at the tissue/material interface are starting to reach clinical application; and an emerging understanding of matrix/cell interactions may soon allow engineered replacement for a range of tissues in the eye.
A basic classification divides materials according to their primary bonding structure into ceramics (ionic bonding), metals (metallic bonding), and polymers (covalent bonding). Modern ophthalmic implants are almost all fabricated from synthetic polymers.
Polymeric materials are composed of long chain molecules (polymers) synthesised from repeat units (monomers) whose chemical character and reactivity determine many bulk properties. Most polymer chains have a covalently bonded backbone of carbon atoms joined to a variety of pendant groups. For siloxanes (“silicone”), an important group of synthetic biomaterials, this backbone consists of alternating atoms of silicone and oxygen. Molecular chains vary in length and are irregularly intertwined, although areas of regular arrangement (crystallinity) may exist. Cross linkage density and the density of secondary bonding further determine bulk properties for a given polymeric material.1
Secondary bonding mechanisms (for example, hydrogen bonds, van der Waals forces) are particularly relevant to biological systems, and are thought to have an important role in modulating protein conditioning—the process by which relatively inert polymeric material surfaces are rendered biologically active by contact with the tissues or body fluids.2
Protein conditioning is partly determined by surface reactivity, which varies …
The objectives of this study were to evaluate the regenerated axon structure at near-terminal locations in the peroneal and tibial branches 1 year following implantation of several tubular devices in a 10-mm gap in the adult rat sciatic nerve and to determine the extent of recovery of selected sensory and motor functions. The devices were collagen and silicone tubes implanted alone or filled with a porous collagen-glycosaminoglycan matrix. Intact contralateral nerves and autografts were used as controls. Nerves were retrieved at 30 and 60 weeks postoperatively for histological evaluation of the number and diameter of regenerated axons proximal and distal to the gap and in the final and peroneal nerve branches, near the termination point. Several functional evaluation methods were employed: gait analysis, pinch test, muscle circumference, and response to electrical stimulation. A notable finding was that the matrix-filled collagen tube group had a significantly greater number of large-diameter myelinated axons (≥6 μm in diameter) in the distal nerve branches than any other group, including the autograft group. These results were consistent with previously reported electrophysiological measurements that showed that the action potential amplitude for the A fibers in the matrix-filled collagen tube group was greater than for the autograft control group. Functional testing revealed the existence of both sensory and motor recovery following peripheral nerve regeneration through all devices; however, the tests employed in this study did not show differences among the groups with regeneration. Electrical stimulation in vivo showed that threshold parameters to elicit muscle twitch were the same for reinnervating and control nerves. The investigation is of importance in showing for the first time the superiority of a specific fully resorbable off-the-shelf device over an autograft for bridging gaps in peripheral nerve, with respect to the near-terminus axonal structure.
The objective of this study was to investigate the contractile behavior of peripheral nerve support cells in collagen-glycosaminoglycan (GAG) matrices in vitro. Contractile fibroblasts (myofibroblasts) are known to participate in wound contraction during healing of selected connective tissues (viz., dermis), but little is known about the activity of non-muscle contractile cells during healing of peripheral nerves. Explants from adult rat sciatic nerves were placed onto collagen-GAG matrix disks and maintained in culture for up to 30 days. Groups of collagen-GAG matrices were tested that differed in average pore diameter and in degree of cross-linking. Cell migration from nerve explants into the matrices was examined, and immunohistochemical staining was used to identify cells expressing a contractile actin isoform (alpha-smooth muscle actin; alpha-SMA) and Schwann cells (S-100). Geometric contraction of matrix disks was quantified every five days as the percent reduction in disk diameter. The amount of contraction of matrix disks was significantly affected by the degree of cross-linking. Cell migration into the matrices and the distribution of cells staining for alpha-SMA or S-100 was not affected by matrix parameters. These studies demonstrate that cells from peripheral nerve explants were capable of adopting a contractile phenotype and causing geometric contraction of matrices in vitro and suggest that contractile processes may be important during nerve wound healing in vivo.
Induced organ regeneration is de novo synthesis of a physiological, or nearly physiological, organ at the same anatomical site as the organ that is being replaced. Regeneration of skin, peripheral nerves and the conjunctiva, described in this chapter, have been accomplished using biologically active scaffolds (regeneration templates) seeded with epithelial cells; devices for regeneration of the first two organs are in clinical use. There is substantial empirical evidence that templates induce regeneration by blocking contraction, the major mechanism for closure of severe wounds in adults. Templates appear to function by interfering with normal myofibroblast function well as by acting as temporary configurational guides for synthesis of new stroma that resembles that of the organ under replacement. The combined evidence supports a theory which predicts that selective blocking of the adult healing response uncovers the latent fetal response to injury and leads to organ regeneration. An independent theory suggests that loss of regenerative potential during the mammalian fetal-adult transition is associated with simultaneous acquisition of individual immunocompetence.
Progress in understanding conditions for optimal peripheral nerve regeneration has been stunted due to lack of standardization of experimental conditions and assays. In this paper we review the large database that has been generated using the Lundborg nerve chamber model and compare various theories for their ability to explain the experimental data. Data were normalized based on systematic use of the critical axon elongation, the gap length at which the probability of axon reconnection between the stumps is just 50%. Use of this criterion has led to a rank-ordering of devices or treatments and has led, in turn, to conclusions about the conditions that facilitate regeneration. Experimental configurations that have maximized facilitation of peripheral nerve regeneration are those in which the tube wall comprised degradable polymers, including collagen and certain synthetic biodegradable polymers, and was cell-permeable rather than protein-permeable. Tube fillings that showed very high regenerative activity were suspensions of Schwann cells, a solution either of acidic or basic fibroblast growth factor, insoluble ECM substrates rather than solutions or gels, polyamide filaments oriented along the tube axis and highly porous, insoluble analogs of the ECM with specific structure and controlled degradation rate. It is suggested that the data are best explained by postulating that the quality of regeneration depends on two critical processes. The first is compression of stumps and regenerating nerve by a thick myofibroblast layer that surrounds these tissues and blocks synthesis of a nerve of large diameter (pressure cuff theory). The second is synthesis of linear columns of Schwann cells that serve as tracks for axon elongation (basement membrane microtube theory). It is concluded that experimental configurations that show high regenerative activity suppress the first process while facilitating the second.
Acute management includes physical examination at the time of injury as a baseline for more detailed evaluations of motor and sensory function. The decision regarding type of nerve suture is based on the extent of the extremity wound and the cause of injury. In the closed extremity injury, spontaneous nerve recovery can be correlated with cause of injury. The evaluation of clinical recovery is based on nerve return and extremity coordination.
Nontoxic, bioresorbable "nerve guide" tubes were used to bridge the transected optic nerves of adult rats. Nerve guides were fabricated as polymers of synthetic poly D,L-lactates with 2% triethyl citrate added as a plasticizer. The local environment was manipulated further by the addition of the proteins collagen, fibrinogen, and anti-Thy-1 antibody to the nerve guide lumens at the time of operation. Neovascular growth through the nerve guide lumens was quantified with the aid of a computer-controlled microscope. Neovascular growth was greater in the nerve guides to which proteins had been added, compared with initially empty nerve guides. These experiments demonstrated the effectiveness of these nerve guide tubes in supporting and directing neovascular growth in the mammalian central nervous system, and suggested that specific alterations of the local environment within the nerve guide lumen can affect the extent of neovascular growth.
Severe nerve injuries may require microsurgical grafting to span a defect. Introduction of graft material into a highly vascular recipient bed is documented to aid in early regeneration of neuronal blood supply.
A silicone rod (SR)-induced fibrovascular sheath was employed to evaluate the regeneration of rat tibial nerve through 2-mmdiameter collagen tubes (CT) or contralateral nerve autografts (AUTO). At first operation, 5 mm of right tibial nerve was resected from 30 retired male breeder Sprague-Dawley rats. Resected nerve was replaced with either a 5 x 2 mm SR or the nerve ends were sutured to the intermuscular fascia.
Four weeks later, animals were repaired by replacing the SR with either a CT or a contralateral AUTO from the left tibial nerve. Three months later, EMG testing was performed, and histologic sections were prepared.
The EMG latency and the size of the compound action potential for sheathed or non-sheathed CT or AUTO were statistically superior to controls at the 95% confidence level. All other intergroup comparisons of latency and action potential size were statistically insignificant. The proportion of nerve fibers traversing the surgical sites was not influenced by the method of repair or by the presence or absence of sheathing. Tubulized repairs most closely resembled unoperated nerves, and autografted repairs had a large diameter, but much fibrosis, whereas controls displayed immaturity and disorganization.
Our observations suggest that there was no difference between repairs performed with or without a vascular pseudosheath. However, CT supported regeneration better than did AUTO repair.
A new peripheral nerve forms across a 10 mm gap within a silicone chamber regeneration model when the distal segment of a transected sciatic nerve, connected to its end organs, is sutured into the distal end of the chamber. We have tested the ability of other tissue inserts to support axonal regeneration in the chamber. When an isolated 2 mm piece of sciatic nerve was sutured into the distal end, fibrin matrix formation, cell immigration and axonal regeneration were identical to those occurring in the control. When the distal nerve insert was replaced with a 2 mm piece of skin or a ligation, a matrix did not form and subsequent cell immigration and axonal regeneration did not occur. When a 2 mm piece of tendon was inserted, a matrix did form at 1 week, but a structure across the gap was observed at later time periods in only 2 out of 7 chambers. The matrix either dissolved before cells could enter the chamber or did not promote cellular immigration and subsequent axonal regeneration. When the distal end was left open, a matrix formed and cells from the reactive tissue outside the chamber entered the matrix and formed a granulation tissue bridge across the gap. This tissue failed to support axonal regeneration; at 3 weeks, axons stopped 1 mm beyond the proximal stump at the interface with the granulation tissue. Thus, matrix formation and a cellular bridge are necessary but not sufficient to ensure regeneration. Successful regeneration across the silicone chamber gap requires humoral and/or cellular contributions available from peripheral nervous tissue and not from the other tested tissues.
Rat spinal cords were subjected to a 200 g/cm force acceleration injury at T10. Ten days later, the cords were totally transected at T10 and the rats separated into two groups: group C (controls) had the spinal cord realigned end-to-end; group X had 3 mm trimmed from proximal and distal cord stumps and a semifluid collagen matrix (CM) bioimplant was inserted in the gap. The CM polymerized to a firm gel at body temperature within 2 h. All rats were maintained 90 days posttransection (dpt). At 90 dpt, they were examined for local spinal cord blood flows, somatosensory evoked potentials, and a neurological evaluation. After killing, the cords were processed for electron and light microscopy and monoamine histofluorescence. The results indicated that CM can support the growth of central neurites, fibroblasts, and an adequate anastomotic network of blood vessels. Control scar tissue does not promote the presence of nerve fibers and blood vessels to the extent observed in the CM. Somatosensory evoked potential early waveforms were present in CM-bioimplanted rats but not in controls. No rat regained walking ability at 90 dpt but muscle tone and strength appeared better in CM-implanted than in control rats. We conclude that a CM bridge can provide a well vascularized, relatively nonhostile environment for central neurites and catecholaminergic axons extending from the proximal spinal cord tissue across the CM bridge and into the distal stump.
The part played by basement membrane in the guidance of peripheral nerve growth in vivo has been assessed by examining the capacity of degenerating mouse muscle to support the regeneration of the cut sciatic and saphenous nerves. Ethanol and formaldehyde-fixed gluteus maximus muscles were implanted around the contralateral cut nerves. The subsequent nerve growth into the degenerating muscle was assessed by silver staining after 3, 4 and 10 days. By 4 days, linear axonal growth was seen, parallel to the length of the muscle fibres, and coinciding with the onset of degeneration of the sarcoplasm. Transverse sections of the 10 day preparations showed that over 90% of linearly growing axons were located inside the remaining sheaths of muscle fibre basement membrane. This relationship was confirmed by electron microscopy of ruthenium red-stained preparations. Both motor and sensory axons were able to grow in this manner, for electrophysiological testing revealed the presence of motor axons from the sciatic nerve, while the saphenous nerve contains only sensory axons. Identical growth was seen at 10 days in muscles caused to degenerate by incubation in distilled water. However, linear growth did not occur in live-innervated and glutaraldehyde-fixed muscles, in which muscle fibre architecture was preserved. It is concluded that basement membrane derived from muscle can promote peripheral nerve regeneration. Furthermore, both motor and sensory axons show a strong preference for growth along its inner surface, the basal lamina.
A bilayer artificial skin composed of a temporary Silastic epidermis and a porous collagen-chondroitn 6-sulfate fibrillar dermis, which is not removed, has been used to physiologically close up to 60% of the body surface following prompt excision of burn wounds in ten patients whose total burn size covered 50--95% body surface area (BSA). Following grafting, the dermal portion is populated with fibroblasts and vessels from the wound bed. The anatomic structure of the artificial dermis resembles normal dermis and serves as a template for the synthesis of new connective tissue and the formation of a "neodermis," while it is slowly biodegraded. This artificial skin has physiologically closed excised burn wounds for periods of time up to 46 days before the Silastic epidermis was removed. At the time of election when donor sites are ready for reharvesting, the Silastic epidermis is removed from the vascularized artificial dermis and replaced with 0.004 autoepidermal graft in sheet or meshed form. Clinical and histologic experience in a relatively short follow-up period (2--16 months) indicates that "neodermis" retains some of the anatomic characteristics and behavior of normal dermis, thus promising improvement in the functional and cosmetic results, as well as providing physiologic function as a skin substitute. The artificial skin is easily sterilized and stored at room temperature, capable of large scale production, and immediately available for grafting, indicating its potential for easy and relatively economic use in the burn patient.
The regeneration of transected mouse sciatic nerves using semipermeable acrylic copolymer tubes to enclose both stumps has been qualitatively assessed from 1 to 30 weeks post-operative. Quantitative morphometric analysis of electron micrograph montages of complete transverse sections of the segment regenerated between stumps has permitted determinations of the percents of total area occupied by the various tissue constituents--blood vessels, epineurium, perineurium, endoneurium, myelinated axon/Schwann cell units, and unmyelinated axon/Schwann cell units. Significant differences were found in the total cross-sectional area of segments regenerated through tubes of 1.0 mm versus 0.5 mm internal diameters. Segments regenerated with the distal stump inserted in the tube contained significantly greater percentages of neural units and were significantly larger at 8 weeks post-operative compared to segments regenerated for 9-10 weeks with the distal stump avulsed. The morphometric method permits rapid quantitation of sizeable electron micrograph montages which at 1300 X permit all types of tissue components, including the unmyelinated axons, to be visualized.
Prompt and long-term closure of full-thickness skin wounds is guinea pigs and humans is achieved by applying a bilayer polymeric
membrane. The membrane comprises a top layer of a silicone elastomer and a bottom layer of a porous cross-linked network of
collagen and glycosaminoglycan. The bottom layer can be seeded with a small number of autologous basal cells before grafting.
No immunosuppression is used and infection, exudation, and rejection are absent. Host tissue utilizes the sterile membrane
as a culture medium to synthesize neoepidermal and neodermal tissue. A functional extension of skin over the entire wound
area is formed in about 4 weeks.
We describe an experimental in vivo system for studying peripheral nerve regeneration, in which the proximal stump of a transected nerve regrows through a transparent silicone chamber toward the distal stump. Physical separation permits examination of the effects of the humoral and/or cellular influences from the distal stump on regenerating fibers before they invade the distal segment itself. A small segment of the rat sciatic nerve was resected, leaving a 6 mm gap which was then encased by a cylindrical silicone chamber. Within the first weeks, a nerve trunk regenerated along the central axis of the chamber bridged the gap between the proximal and distal stumps. When the distal nerve stump was omitted from the distal opening of the chamber, only a thin structure with a few small-caliber fibers extended across the gap. In each instance regenerating nerve appeared as a cord-like structure completely surrounded by clear fluid, a feature which permits easy collection of the extracellular fluid for analysis of its chemical properties and biological activity. This feature also allows in vivo manipulation of the humoral environment in which nerve regeneration occurs.
The range of growth-promoting influences from a distal nerve stump on a regenerating proximal stump was determined using an experimental system in which a gap between cross-anastomosed rat sciatic nerves was encased by a cylindrical silicone chamber. Two arrangements were examined after 1 month in situ: A proximal-distal (PD) system in which both proximal and distal stumps were introduced into the ends of the chamber, and a proximal-open (PO) system in which the distal stump was omitted. When the gap was 6 mm long, a regenerated nerve extended all the way through the chamber in both the PD and PO systems. When the gap was increased to 10 mm, a similar regrowth occurred in the PD chamber, whereas in the PO chamber proximal regrowth was partial or nonexistent. When the gap was increased to 15 mm, no regeneration occurred, even in the presence of the distal stump. These observations confirm that the distal stump influences proximal regeneration and indicate that this influence can act only over a limited distance or volume. Such an influence could consist of humoral agents which support nerve growth and/or outgrowth from the distal stump.
Detailed methodology is described for the reproducible preparation of collagen--glycosaminoglycan (GAG) membranes with known chemical composition. These membranes have been used to cover satisfactorily large experimental full-thickness skin wounds in guinea pigs over the past few years. Such membranes have effectively protected these wounds from infection and fluid loss for over 25 days without rejection and without requiring change or other invasive manipulation. When appropriately designed for the purpose, the membranes have also strongly retarded wound contraction and have become replaced by newly synthesized, stable connective tissue. In our work, purified, fully native collagen from two mammalian sources is precipitated from acid dispersion by addition of chondroitin 6-sulfate. The relative amount of GAG in the coprecipitate varies with the amount of GAG added and with the pH. Since coprecipitated GAG is generally eluted from collagen fibers by physiological fluids, control of the chemical composition of membranes is arrived at by crosslinking the collagen--GAG ionic complex with glutaraldehyde, or, alternately, by use of high-temperature vacuum dehydration. Appropriate use of the crosslinking treatment allows separate study of changes in membrane composition due to elution of GAG by extracellular fluid in animal studies from changes in composition due to enzymatic degradation of the grafted or implanted membrane in these studies. Exhaustive in vitro elution studies extending up to 20 days showed that these crosslinking treatments insolubilize in an apparently permanent manner a fraction of the ionically complexed GAG, although it could not be directly confirmed that glutaraldehyde treatment covalently crosslinks GAG to collagen. By contrast, the available evidence suggests strongly that high-temperature vacuum dehydration leads to formation of chemical bonds between collagen and GAG. Procedures are described for control of insolubilized and "free" GAG in these membranes as well as for control of the molecular weight between crosslinks (Mc). The insolubilized GAG can be controlled in the range 0.5--10 wt. % while "free" GAG can be independently controlled up to at least 25 wt. %; Mc can be controlled in the range 2500--40,000. Studies by infrared spectroscopy have shown that treatment of collagen--GAG membranes by glutaraldehyde or under high-temperature vacuum does not alter the configuration of the collagen triple helix in the membranes. Neither do these treatments modify the native banding pattern of collagen as viewed by electron microscopy. Collagen--GAG membranes appear to be useful as chemically well-characterized, solid macromolecular probes of biomaterial--tissue interactions.
Several methods are compared for preparing collagen-glycosaminoglycan (GAG) membranes of high or low porosity. Collagen-GAG membranes have been used to cover satisfactorily large experimental full-thickness skin wounds in guinea pigs over the past few years. Methods studied as means for controlling pore size are confined to purely physical processes which do not require use of additives or chemical reagents to form the porous membrane. We find that membranes, initially swollen in distilled water or saline, shrink linearly to no less than 94% of original dimension after freeze drying; to 75% after critical point drying (from CO2, following water-ethanol exchange); and to 41% of original dimension following air drying from the swollen state. Scanning electron microscopic study of the pore structure resulting from eah drying procedure confirms our major conclusion: A carefully designed freeze drying process, two variants of which are described in detail, yields membranes with the highest mean pore size, as measured by quantitative stereological procedures. Critical point drying gave significantly more shrinkage and a lower mean pore size than either one of the two freeze drying procedures used.