Invented by David F. Muir, University of Florida Research Foundation Inc
The University of Florida Research Foundation Inc invention works as followsThe subject invention relates to compositions, methods and devices for promoting the repair of damaged tissue by using nerve grafts. The compositions of the invention can be used to restore continuity of nerve interrupted due to disease, trauma or surgical procedures. The compositions of the invention include one or more chondroitin sulfate protoglycan degrading enzymes. These enzymes promote axonal penetrating into damaged nerve tissue, and nerve graft. The invention also relates to methods of promoting the repair of damaged nervous tissue by using the compositions, and nerve tissue that has been treated in accordance with such methods. The invention includes solutions for storing nerve tissue.
Background for Materials for nerve grafting: Materials and methods
Peripheral Nerve Injuries are a major cause of chronic disability.” When severed axons cannot reestablish continuity, they can cause painful neuromas. The ability of nerves to regenerate is dependent on the regenerating fibers and their axonal sprouts making proper contact with the severed segment (and Schwann cells basal laminae). If the regenerating axons do not enter the base lamina and cross the gap, they will degenerate, leading to neuronal death and muscle atrophy (Fawcett, J.W. et al., 1990).  Annu Rev Neurosci 13:43-60).
Briefly a nerve carries peripheral processes (or the axons of neurons). The neuronal cells are located in the spinal column (motor neurons), the ganglia along the vertebral columns (spinal sensor ganglia), or the ganglia that cover the entire body (autonomic, enteric, and ganglial ganglia). A nerve is made up of axons and Schwann cells, as well as extensive connective tissue coverings (Dagum AB  J Hand Ther 11, 111-117). The epineurium is a layer of connective tissue made up of collagen that protects the fascicles against external pressure. It also surrounds the perineurium. Perineurium surrounds fascicles. Endothelial cell in endoneurial vessels act as the barrier between the blood and nerves. The endoneurium is located inside the perineurium. It is made up of collagenous tissue and surrounds Schwann cells, axons and Schwann cell axons. A fascicular group is made up of two or three fascicles, which are surrounded by epineurium and perineurium. The topography is constant in distance, and a grouping of fascicles can be either motor or sensory. The neuron is made up of two parts: a cell body (soma) and a long axon.
In nerve injuries that cause axonal disruption but where the endoneurial layer remains intact (e.g. crush injury), the axons will regenerate in their original lamina basal and complete recovery is possible. The axonal regeneration may be severely impaired after nerve transection, and the surgical repair depends on the realignment described above. (Dagum AB  J Hand Ther 11, 111-117). The primary way to deal with nerve transection is epineurial coaptation. The extent of regeneration can vary greatly and at best only partial function is expected (Terzis, J.K. et al.  Hampton Press, Norfolk, The Peripheral Neuron: Structure, Function and Reconstruction. The full restoration of function following repair of nerve transsection is not possible due to the fine microstructure and inability of axons-to-axons coaptation.
Nerve grafting may be warranted in cases of nerve ablation, but it presents some practical challenges.” Over the years, different nerve graft options have been explored. Allogenic nerve grafts are currently viewed as an alternative. The availability of donor grafts is limited, but the importance of viable cells in nerve grafts could be less significant. The nerve sheath contains the necessary scaffolding and adhesives cues for axonal regrowth. Significant regeneration has also been achieved with acellular nerve grafts, e.g. freeze-killed nerve grafts.  Brain Res 288:61-75; Hall S M  Neuropathol Appl Neurobiol 12:401-414; Gulati A K  J Neurosurg 68:117-123; Nadim W et al.  Neuropathol Appl Neurobiol 16:411-421). The resident antigen-presenting cell (e.g. Schwann cells fibroblasts endothelial, Schwann, etc.) must be killed. The immunogenicity is greatly reduced. The use of acellular grafts reduces or even eliminates host-graft immune rejection (Evans, P. J. et al.  Prog Neurobiol 43:187-233; Evans P J et al.  Muscle Nerve 21, 1507-1522). These characteristics offer great promise for the use freeze-killed allogenic and xenogenic grafts. The absence of viable cell prevents the degeneration of nerve tissue and the subsequent remodeling, which appears to encourage the regeneration process (Bedi KS et. al.  Eur J Neurosci 4:193-200; Danielsen N et al.  Brain Res 666:250-254).
Laminin represents an adhesive stimulus that is necessary for successful axonal regrowth (Wang, G.Y. et al.  Brain Res. 570:116-125. Normal nerve, even though it is rich in laminin (uninjured), remains inhibitory or refractory of axonal development. (Langley J. N.  J Physiol, 31:365-391. Brown M. C. et.  Eur J Neurosci 6:420-428). This suggests that laminin’s growth-promoting function is suppressed within a normal nervous environment, and that this activity must be reactivated in nerve regeneration and degeneration.
Normal peripheral nervous is a poor substrate for axonal development (Zuo, J.  J Neurobiol 34: 41-54; Bedi K S et al.  Eur J Neurosci 4: 193-200). Experimental results show that the laminin in normal nerve basal layers is not accessible by regenerating axons sprouts (Zuo, J. and al.).  J Neurosci 18: 5203-5211; Ferguson T A, and D. Muir  Mol Cell Neurosci 16: 157-167; Agius E. et al.  J Neurosci 18: 328-338). After injury, the segment that is severed (distal from the injury) goes through a degenerative process which initiates extensive remodeling. In nerve degeneration caused by injury, the severed myelin-sheath fragments, along with the debris, are removed through phagocytosis. The sheath structure and the basal lamina remain intact despite this degeneration. Schwann cells multiply and prepare nerves for regrowth. The entire process of nerve degeneration, including the remodeling, is referred to. The distal segment of the nerve is positively modified by nerve injury. Experiments have shown that degenerated nerves are more able to promote axon regeneration than normal nerves (Bedi, K. S. et. al.  Eur J Neurosci 4: 193-200; Danielsen N J et al.  Brain Res 666: 250-254; Agius E et al.  J Neurosci 18: 328-338). The degenerative process is thought to be a result of mechanisms that transform normal nerves from a state that suppresses growth to one that encourages it (Salonen, V.J. et al.  J Neurocytol 16: 713-720; Danielsen N et al.  Brain Res 681: 105-108).
Axon disruption is the cause of loss of function in nerve injuries. Axons can be very fragile and thin, and even the slightest injury can result in a severe response (axotomy). Axotomy occurs when the axons distal to the injury die and degenerate. Axonotmesis is the least problematic nerve injury. It involves axotomy, but the continuity between the nerve sheaths is preserved. Axons regenerate in the case of an axonotmesis because the laminae are still intact. Axonal sprouts from the proximal stump of the nerve must first locate and then reach the Schwann cell laminae at the distal segment to successfully regenerate severed peripheral nervous systems. It is believed that this crucial requirement contributes to the relatively poor recovery after nerve transection compared with crush injury. The nerve can be partially or completely severed in nerve transection. Transection injuries occur when both the axons as well as the sheaths of the nerve are severed. This disrupts the continuity of a nerve and the necessary guidance mechanisms for axon regrowth. For regrowth of nerve axons, surgical coaptation (neurorrhaphy), to restore the continuity of the nerve elements is required. The misalignment between proximal elements and distal ones complicates axonal regeneration after nerve transection. The nerve structure is affected even in cases of clean transection with a sharp instrument. The swelling and axoplasmic flow from the cut ends can cause a mushrooming, which interferes in the accurate coaptation of the scaffolding at the base lamina. Axon-to -axon alignment remains an idealistic target despite improvements in fascicular alignement achieved by microsurgical techniques. Due to the relative abundance of connective tissue and the small size of axons, most axonal sprouts that emerge from the proximal tibial stump following surgical coaptation will first encounter a nonpermissive substrate rich in inhibitory chondroitin sulfate proglycan. This could explain the latency and irregular regeneration that are associated with peripheral nerve repair. Evidence suggests that CSPGs bind and inhibit laminin’s growth-promoting properties, and that CSPGs are degraded in the degenerative processes after injury. The process by which CSPGs become inactive can therefore explain why nerve regeneration is so important. Recently, it was discovered that peripheral nerves contain abundant CSPG which inhibits growth-promoting activities of endoneurial linmin (Zuo J et. al. [1998a] J Neurobiol 34:41-54). The neurite inhibiting CSPGs, which are abundant in endoneurial tissue surrounding Schwann cells basal laminae are rapidly upregulated following nerve injury. [1995a] Eur J Neurosci 7:805-814; Braunewell K H et al. [1995b] Eur J Neurosci 7:792-804). In consequence, misalignment in nerve microstructure after injury and repair forces regenerating sprouts to navigate nonpermissive tissue which can severely restrict their access to the basal laminae of the distal nerve. Recent research has supported the conclusion that certain CSPG degrading enzymes are a mechanism through which growth-promoting laminin properties can be restored in degenerating nerve. (Zuo, J. et al. [1998b] J Neurosci 18:5203-5211; Ferguson T A et al.  Mol Cell Neurosci 16:157-167). This process can also be improved by applying CSPG-degrading enzymatic agents to the nerve injury site and nerve grafts in order to enhance regeneration (Zuo, J. et. al.  Exp Neurol 176: 221-228; Krekoski C A et al.  J Neurosci 21: 6206-6213). One CSPG-degrading bacterial enzyme, chondroitinase ABC (Zuo J et. al.), is especially effective at degrading the disaccharide-side-chains in CSPG. [1998a] J Neurobiol 34:41-54). The matrix metalloproteinase MMP-2 and MMP-9 are also involved in the degradation of CSPG’s core protein (Ferguson, T.A.  Mol Cell Neurosci 16: 157-167).
The ability of chondroitinase ABC to promote growth in nervous tissue is attributed to CSPG degrading (Zuo, J. et al.  Exp Neurol 154:654-662; Ferguson T A et al.  Mol Cell Neurosci 16:157-167). It has also been demonstrated that chondroitinase-ABC treatment does not disrupt the nerve sheath organisation or displace laminin in the Schwann cells basal lamina. (Krekoski, C. A. et. al.  J Neurosci 21:6206-6213).
In nerve transection repair, degradation of inhibitory CSPG eliminated a major barrier to regenerating axonal stems and resulted into more robust and uniform growing into the distal nervous (Krekoski A et. al.  J Neurosci 21:6206-6213).
It was shown that degenerated nerves have an increased ability of supporting axonal development (Giannini et al.  J Neuropathol Exp Neurol 49:550-563; Hasan N et al.  J Anat 189:293-302). Since axonal regeneration can be improved in acellular grafts made from predegenerated nervous tissue, the effects of degeneration may also be due to modifications in the nerve basal layer (Danielsen et al.  Brain Res 681:105-108). The Schwann basal lamina is structurally intact throughout the degenerative process.
Animal models showed that grafts from nerves which have been predegenerated in-vivo were much more effective at supporting nerve regeneration (Danielsen, N. et al.).  Brain Res 681:105-108). The procedure to create pre-degenerated human nerves is not practical (i.e. nerve injury followed by survival in vivo for tissue degeneration).
The release of proteolytic proteins by Schwann cells, neurons and macrophages invaders is responsible for peripheral nerve degeneration. The modification of matrix metalloproteinase activities (MMP) after injury implicates MMP-2, and MMP-9 remodeling the extracellular matrix in nerve regeneration and degeneration (La Fleur et.al.  J Exp Med 184:2311-2326; Kherif et al.  Neuropathol Appl Neurobiol 24:309-319; Ferguson et al.  Mol Cell Neurosci 16:157-167). MMP-9 is primarily expressed at the site and immediately following injury of the peripheral nerve. MMP-9 is correlated with the breakdown in the blood-nerve barriers, accumulation of granulocytes, and invasion of macrophages. (Shubayev and al.  Brain Res 855:83-89; Siebert et al.  J Neuropathol Exp Neurol 60:85-93). Taskinen and colleagues found that the majority of evidence indicates that hematogenic cell contribute to an increase in MMP-9 activity.  Acta Neuropathol (Berl) 93:252-259). MMP-2, on the other hand is constitutively expressed by Schwann cell in normal peripheral nerves (Yamada and al.  Acta Neuropathol (Berl) 89:199-203). MMP-2 is expressed more than a week after the injury and the latent enzyme is converted into its active form.  Mol Cell Neurosci 16:157-167).
In vitro degeneration leads to a significant increase in neurite-promoting activities of nerve explants. The addition of MMP inhibitors blocks this increase, and the increase in net gelatinolytic activities (as demonstrated by in situ zymography) is also blocked. In cultured nerve explants, the increase in neurite-promoting activities occurs quickly and parallel to the upregulation and activation MMP-2. The initial effect of degeneration in vivo only suppresses normal nerve’s already low level of neurite-promoting activity. During this time, there is no change to MMP-2 activation or expression in vivo. “The neurite-promoting activities of transected neurons increase in vivo over time, and this is accompanied by a surge of MMP-2 activation and expression (Ferguson & Muir 2000, Mol Cell Neurosci 16, 157-167, Shubayev & Myers 2000, Brain Res 855, 83-89).
In vitro tests indicate that predegenerated nerve segments in vivo are more likely to promote neurites than normal nerve segments (Bedi and al.  Eur J Neurosci 4:193-200; Agius et al.  J Neurosci 18:328-338; Ferguson et al.  Mol Cell Neurosci 16:157-167). In vivo tests of predegenerated nerves grafts produced contradictory results, particularly when using cellular nerve grafts.  J Hand Surg [Am] 4:42-47; Danielsen et al.  Brain Res 666:250-254; Hasan et al.  J Anat 189(Pt 2):293-302). However, predegeneration seems to be especially advantageous for the enhancement regeneration into acellular transplants (Ochi and al.  Exp Neurol 128:216-225; Danielsen et al.  Brain Res 681:105-108). This suggests that in degeneration, molecular and cellular mechanisms enhance the growth-promoting abilities of the basal layer, which retains its ability to stimulate regeneration after cellular elements are killed. Predegeneration in vitro results in an increase in growth-promoting abilities of acellular grafts. This was easily demonstrated by the cryoculture models and grafting methods used in the present invention. Acellular nerve grafting has a significant latency for axonal regeneration. (Danielsen and al.  Brain Res 681:105-108).
Much of the research in nerve explant cultures and nerve grafts preservation has been focused on cold storage of segments of nerve. Cold storage methods are intended to preserve nerves under minimal, ischemic conditions to suppress cellular activity and proteolytic activity. Levi et al. (Levi A et al.  Glia 10, 121-131) showed that the viability of cells decreased significantly after a week, and only a small number of viable Schwann cell remained after three weeks. Subsequently, Lassner et al. (Lassner et al. J Reconstr Microsurg, 11:447-453 (1995), reported that culture mediums (DMEM rather than Cold Storage Solution), have a positive impact on the viability of Schwann cells and the regenerative capacity of nerve grafts in cold ischemic storage conditions. Even though cold storage is not optimal for promoting growth in nerve grafts it does decrease cell viability and immunogenicity, as well as the concern of immunorejection. (Evans et.al.  Muscle Nerve 21, 1507-1522. This is why nerve allografts that have been frozen-killed and stored for a long time are more likely to regenerate than those that have not. (Evans, et. al. Microsurgery (1999) 19:115-127).
Accordingly, there is a need for a low-risk adjunctive therapy that can improve the results of conventional nerve repairs.
The subject invention relates to compositions and methods that promote the repair of nervous tissue. In a preferred form, the compositions of this invention contain chondroitin sulfate protoglycan degrading enzymes. In one embodiment, a subject invention composition comprises a CSPG degrading enzyme chosen from the group consisting chondroitinase hyaluronidase matrix metalloproteinase MMP, or combinations thereof. In a second embodiment, a subject invention composition comprises a CSPG degrading enzyme chosen from the group consisting chondroitinase ABC (chondroitinase ABC), chondroitinase B, chondroitinase D, chondroitinase E, hyaluronidase MMP-2 and MMP-9 or combinations thereof.
The present invention also relates to methods for promoting the repair of damaged nervous tissue in an animal or human. The present invention comprises methods of administering one or multiple CSPG-degrading enzymatic agents to a damaged nerve tissue, a coaptation or a repair. The present invention increases the ability of regenerating nerve axons, to cross the interface between the nerve and the nerve graft or the nerve-nerve. It also enhances axonal development within the basal layer scaffold. The degradation of inhibitory CSPG allows axons to have greater access to Schwann cells basal lamina in the nerve. This increases the number of axons which successfully penetrate damaged tissue or implanted grafts. It may also enable the proper routing of axons sprouts, leading to improvements in function.
The present invention also relates to methods for preparing nerve grafts using CSP-degrading enzymatic agents. The nerve graft, whether it is allogenic or xenogenic, is preferably fresh and has not degenerated. It is then treated with CSPG enzymes before or after freezing. The graft may be implanted alive or frozen to kill the cells. In one embodiment, nerve tissue is rendered non-cellular following treatment. “In a preferred embodiment the nerve tissue can be rendered acellular through freeze-killing.
The present invention also relates to methods for culturing nerve tissue that is fresh or temporarily preserved for transport, for implantation into a person or animal as a nerve transplant. The nerve tissue is preferably harvested from a human or an animal donor, and then cultured in physiological conditions which allow the tissue to degenerate, remodel and activate the basal layer by endogenous mechanisms. In one embodiment, after culturing the nerve tissue/graft becomes acellular. “In a preferred embodiment the nerve tissue/graft can be rendered acellular through freeze-killing.
The present invention also relates to methods for providing nerve grafts that can be implanted into animals or humans. Preferably the cross-sectional properties of the donor tissue are similar to those of the nerve tissues at the implantation sites.
Click here to view the patent on Google Patents.