JP2003505205A - Cartilage or bone matrix as a nucleic acid delivery vehicle - Google Patents

Abstract

  (57) [Summary] The present invention relates to tissues including, but not limited to, bone and cartilage grafts as carrier matrices that deliver biologically active nucleic acids to those sites to be transplanted. After transplantation, the nucleic acid bound to the graft is taken up and expressed by cells at the graft site, and produces growth factors, regenerative factors, and the like encoded by the nucleic acid. Thus, for example, where the nucleic acid encodes an osteogenic protein or the like, the rate of osteoinduction, induction of healing, and cartilage without further production, purification, isolation and delivery of protein or peptide growth factors The rate of formation or repair or repair is promoted.

Description

Detailed Description of the Invention

FIELD OF THE INVENTION The present invention relates to implants of bone, cartilage, and other tissues as vehicles for delivering nucleic acid compositions. In a particular application of the invention, bone and cartilage implants are treated with a nucleic acid encoding an osteogenic (osteogenic or osteoconductive) gene product. Such gene products include, but are not limited to, osteogenic proteins, cartilage-derived osteogenic proteins, growth factors, and combinations thereof.

BACKGROUND OF THE INVENTION In the orthopedic field, situations where material implantation is required to provide support or replace damaged or diseased bone or cartilage tissue. There is often Traditionally, such implants have been made of titanium or other relatively inert metals or synthetic polymeric materials. Second recovered from first anatomical position
The autologous bone re-implanted in the anatomical position was also left to the discretion of the operating physician. However, even if such a method is effective in the second anatomical position,
It is less than ideal because it introduces morbidity to the first anatomical location. Using allograft bones (from another individual of the same species) or xenograft bones (from another species) to improve the reliability of the technology to remove potentially pathogenic organisms The higher the T, the higher the engraftment rate of cartilage or other material.

Performing spinal fusion results in painful spinal movements and structural abnormalities, trauma instability, degenerative instability, and post-incision iatrogenic instability. Stabilization of the spinal column results in disability. The fusion or arthrodesis is completed with the formation of a bone bridge between adjacent moving parts. Bone bridges in the disc space may be formed anteriorly between adjacent vertebral bodies and posteriorly with continuous transverse processes, laminae, or other posterior surfaces of the vertebrae.

Bone bridges or fusion masses are made biologically by bodies with skeletal damage. By utilizing such a normal bone healing reaction, the operator reproduces the spinal cord injury condition along the fusion site to enable bone healing, thereby inducing abnormal fusion between spinal segments. Successful fusion requires the presence of osteogenic or osteogenic cells, proper blood supply, adequate inflammatory response, and proper preparation of local bone. Such a biological environment is usually provided by debridement (a procedure in which the outer cortical bone is removed to expose bloody cancellous bone) and surgery to accumulate an appropriate amount of high quality implant material.

Fusion or arthrodesis may be performed to treat disk-related disorders. The intervertebral disc is located between the end plates of adjacent vertebrae, stabilizes the spine, distributes forces between the vertebrae, and serves as a cushion to support the vertebral body. A normal intervertebral disc contains a semigelatinous substance, the nucleus pulposus, which is confined by an outer wrapping in a fibrous ring (called the fibrous ring). In a healthy, non-injured spine, the annulus fibrosus prevents the nucleus pulposus from projecting outward from the disc space. The spinal disc may be displaced or damaged due to trauma, disease, or aging. When the annulus fibrosus ruptures, the nucleus pulposus projects into the spinal canal, resulting in what is commonly called an intervertebral disc hernia. The extruded nucleus pulposus compresses the spinal nerves, which may eventually result in neuropathy, pain, numbness, weakness, and paralysis. The intervertebral disc may also deteriorate due to normal aging processes or diseases. When the intervertebral disc is dehydrated and becomes stiff, the space occupied by the intervertebral disc becomes low, the spinal column becomes unstable, and mobility may be reduced, causing pain.

Sometimes, the only way to alleviate the symptoms of such a condition is a discectomy, ie surgical removal of some or all of the disc to fuse adjacent vertebrae. The space occupied by the disc collapses, except for damaged or unhealthy discs. When the disc space collapses, in addition to severe pain,
Spinal destabilization, joint mechanics, premature arthritis, or neuropathy may occur. Maintaining the disc space for pain relief from discectomy and arthrodesis,
And a final fusion of the affected moving parts is required. Bone grafts are often used to fill the space between discs, prevent collapse of the disc space, and promote fusion of adjacent vertebrae beyond the disc space. Early techniques simply placed bone material between adjacent vertebrae (typically on the posterior surface of the vertebrae) and bridged the affected vertebrae with plates or rods to stabilize the spine. . After the fusion was observed, the metallic material that kept the stability of the relevant part became unnecessary and was left as a foreign substance permanently. In addition, the surgical method required for implanting the rod or plate to stabilize the surface during fusion requires a long time and is often complicated. Therefore, it was determined that the optimal solution for stabilizing the removed disc space was to fuse the vertebrae between the individual endplates, preferably without the need for anterior or posterior plate placement. .

Many have developed an acceptable intervertebral disc implant that can be used to replace a damaged disc and maintain the stability of the disc space between adjacent vertebrae, at least until arthrodesis is complete. Has been attempted. To be successful, the implant must provide a temporary support and allow bone ingrowth. Successful discectomy and fusion methods require the bone to grow in close proximity to form a mass. This is because the implants are unlikely to withstand the repeated compressive loads on the spine over the life of the patient. Many attempts to restore the intervertebral disc space after removal of the disc have used metallic devices. U.S. Pat. No. 4,878,915 to Brantigan discloses a solid metal plug. US Patent No. 5,044,1 by Ray
04, 5,026,373, and 4,961,740, 5,015,247 by Michelson, and 4,820,305 by Harms et al., US Pat. No. 5,147,402 by Bohler et al., And by Brantigan. 5th, 1st
No. 92,327 discloses a hollow metal cage structure. Unfortunately, due to the hard material, some metal implants provide stress shielding to the bone graft, increasing the time required for fusion, or when the bone graft is resorbed inside the cage. There is. When implanting a metal implant between the vertebrae, fusion may be delayed and the device may sink into the bone. Metallic devices are also foreign objects that are never completely incorporated into the fused mass.

Various bone grafts and bone graft substitutes have also been used to promote bone formation and to avoid the disadvantages of metallic grafts. The reason why autografts are often preferred is that they have osteogenic potential. Both allografts and autografts are biomaterials that replace the patient's own bone after a period of creeping replacement. Unlike metal implants, bone implants almost disappear over time, but metal implants remain longer than their useful life. Bone grafts have similar elastic moduli to the surrounding bone, thus avoiding stress shielding. The stiffness values of commonly used implant materials greatly exceed both stiffness values of cortical and cancellous bone. The titanium alloy has a stiffness value of 114 GPa and the 316L stainless steel has a stiffness value of 193 GPa. On the other hand, the stiffness value of cortical bone is about 17 Gpa. The use of bone as a graft also allows for excellent post-operative image scanning. This is because there is no scattering like a metal implant on CT or MRI image scans.

Various implants have been made from bone or graft replacement materials to fill the intervertebral disc space after removal of the disc. For example, the Croward dowel is one of the annular implants made by piercing the plug of an iliac allograft or autograft. Croward dowels are bicortical with a porous cancellous bone between the two cortical surfaces. Such nails have relatively poor biomechanical properties,
Especially, the compressive strength is small. Therefore, the Croward type dowel is unsuitable as a spacer for the intervertebral disc space when internal fixation is not performed, because the Croward type dowel may collapse before being fused under the condition where a heavy load is repeatedly applied to the spine. Bone dowels with great biomechanical properties are manufactured by Regeneration Technologie.
s, Inc. (RTI), 1 Innovation Drive, Alachua, Florida 32615) and is patented (see US Pat. No. 5,814,084). Unicortical made from the same type of femoral or tibial condyles
You can use the doug. RTI has also developed cortical bone dowels of the diaphysis with excellent mechanical properties. This is the basis of the 5,814,084 patent (the so-called 814 patent). The dowel also has the added benefit of having a naturally occurring lumen formed from the existing medullary canal of the donor long bone. The lumen can be filled with an osteogenic material such as bone or bioceramics.

Unfortunately, however, there are some difficulties in using bone grafts. Autologous transplants can only be performed in a limited number. Additional surgery increases the risk of infection and blood loss, and may compromise structural integrity at the donor site. In addition, some patients are dissatisfied with the fact that surgery to retrieve the graft results in short-term and long-term pain compared to fusion surgery. Allograft material recovered from allogeneic donors is readily available. However, since allogeneic bone does not have the ability to induce bone by itself, it may remain on a temporary support. The slower fusion rates seen with allograft bones can collapse the disc space before fusion is complete. Allografts and autografts have other drawbacks. The implant alone does not provide the necessary stabilization in carrying the load on the spine. Although internal fixation can solve this problem, it has the inherent drawbacks of requiring more complicated surgery and the use of metal fixation devices. The operator is also often required to repeatedly remove the implant material to maintain the correct size and to fill and stabilize the disc space. Such a trial and error approach increases the time required for surgery. In addition, the implant material typically has a smooth surface that provides the optimum frictional force for fitting between adjacent vertebrae. Slip of the implant can cause damage to the nervous and vascular systems and can collapse the disc space. Even where slippage does not occur, small movements at the implant / fusion interface can impair the healing process required for fusion.

[0011] There have been several attempts to develop bone graft substitutes that avoid the drawbacks while retaining the benefits of metal and bone grafts. Unilab, for example, sells various spinal implants composed of hydroxyapatite and bovine collagen. In each case, it has heretofore been extremely difficult or impossible to develop implants having the biomechanical properties of metals and the biological properties of bones without any of the disadvantages.

Based on these difficulties, studies have been conducted on physiologically active substances that regulate a complex cascade of cellular phenomena involved in bone repair. Such substances include bone morphogenetic proteins used as alternative or additional implant materials. Bone morphogenetic protein (BMP), which is one of a class of osteoinductive factors derived from a bone matrix, can induce osteogenesis when implanted at a fracture site or a bone portion of a surgical target. Recombinant bone morphogenetic protein 2 (rhBMP-2) has been reported to be effective in bone regeneration in skeletal defects in multiple animal models. The use of such proteins requires the design of appropriate carriers and fusion spacers.

Due to the need for safe bone graft materials, bone graft substitutes such as bioceramics have received much attention in recent years. In such attempts to develop alternatives to bone grafts, efforts have been made to avoid the drawbacks of metallic and bone grafts while retaining the benefits of both. Calcium phosphate ceramics are biocompatible and show no infection or immunological problems with allograft materials. Ceramics can be prepared in any amount that is of great benefit compared to the bone material used for allografts as well as autografts. In addition, bioceramics have osteoconductivity and promote bone production at bone sites. Bioceramics provide a porous matrix that further promotes new bone growth. Unfortunately, ceramic implants typically do not have the strength to support the high loads on the spine, requiring alternative fixation methods for fusion.

Among the calcium phosphate ceramics, hydroxyapatite (HA) and tricalcium phosphate (TCP) ceramics are very often used for bone transplantation. Hydroxyapatite is chemically similar to inorganic bone material and is biocompatible with bone. On the other hand, the decomposition of hydroxyapatite is slow.
Beta-tricalcium phosphate is rapidly degraded in vivo and is so brittle that it cannot serve as a support under repeated loading of the spine before fusion. Therefore, the development of implants with the biomechanical properties of metals and the biological properties of bones without any of the disadvantages has been extremely difficult or impossible.

Recently, it has become clear that natural bone minerals do not actually resemble the chemistry and structure of hydroxyapatite as much as previously thought (Spector, 21 Clinics in Plastic). Surgery 437-444, 1994, the entire text of which is incorporated herein by reference). Natural bone minerals include carbonate, magnesium, sodium, hydrogen phosphate, and trace elements. The crystal structure of bone mineral is also different from that of hyaluronan. Other details regarding bone chemistry are described in US Pat. No. 4,882,149 by Spector. Mimicking bone chemistry and microstructure is important in terms of obtaining favorable elastic moduli and resorption rates.

Several attempts have been made to make materials that approximate the microstructure of bone. Also, some methods of removing organic substances from bone to produce bone mineral have been reported. Some materials have been used as drug carriers as described, for example, in US Pat. No. 5,417,975. US Patent No. 4 by Spector
, 882,149 discloses bone mineral materials free of fat and bone proteins. This patent describes a powdery, brittle radiopaque material that can be used to deliver bone growth proteins. Although the minerals in the Specter patent are believed to be closer to natural bone minerals than the synthetic calcium phosphate ceramics, they do not have the property of subject shape with biological characteristics. Miyata
U.S. Pat. Nos. 4,314,380 and 5,573,771 by ta) et al. disclose the addition of collagen or gelatin to bone minerals. However, it is unclear how to make these materials resemble natural bone structures. This is because all proteins break their crystal structure when removed from the bone to be treated. In Urist et al. (110 Arch Surg. 416, 1975), a chemically sterilized, antigen-extracted, autodigested autograft believed to maintain the ability of this material to form. Is described. WO 98/56433 by McKay, published December 17, 1998, which disclosed bone graft complexes and spacers containing bone, "removed bound non-collagenous bone proteins. Treated to do so, followed by a combination of full immersion in bone growth factor. However, this publication makes no mention or suggestion of the use of the osteogenic gene product encoded by the nucleic acid. US Pat. No. 5,763,416 discloses a matrix that is contacted with osteoprogenitor cells within the osteoprogenitor tissue site after treatment with nucleic acid. This patent does not describe or suggest the use of bone or cartilage as a nucleic acid carrier matrix.

WO 97/38729 discloses a matrix other than bone soaked in nucleic acid for the purpose of inducing wound healing. International Publication No. 95/22611
U.S. Pat. No. 5,763,416 discloses a nucleic acid treated bone compatible matrix other than bone or cartilage for the purpose of transferring nucleic acid to a bone graft site.

WO 99/06563 discloses a method of transferring a specific nucleic acid contained in a carrier other than bone or cartilage to a bone graft site.

US Pat. No. 5,899,936 describes a method of removing cells from a harvested tissue and then repopulating the tissue with cells of the desired type. However, this patent does not describe or suggest a bone graft as a matrix for delivering nucleic acids.

US Pat. No. 5,854,207 describes the combined use of bone morphogenetic proteins and stimulators (with an optionally desalted bone matrix implant). However, this patent does not describe or suggest methods of delivering nucleic acids encoding bone morphogenetic proteins that bind to the bone matrix for the purpose of inducing bone or cartilage growth or repair.

Accordingly, the present invention, which provides a composition of a bone or cartilage matrix as a vehicle for nucleic acid delivery, presents a substantial difficulty in inducing bone or cartilage repair or formation using proteins. It is met by the delivery of the encoding nucleic acid and thus meets the needs previously existing in the art.

According to one aspect of the Summary of the Invention invention, the composition of the graft bone or cartilage, spinal spacer, and a variety of other bone or cartilage grafts, cartilage or bone growth factors or repair Includes expressible nucleic acids that encode factors. One aspect of the present invention provides a reduced antigenicity bone (RAB) in combination with a nucleic acid encoding a cartilage or bone inducer group (osteogenic factor or chondrogenic factor). To do. In another aspect of the invention, a composition of the invention is prepared, after preparation, in the spinal column, or in any number of tissues, to replace injured, diseased, or tissue that would otherwise be compromised. Used in other biological locations.

In another aspect of the invention, there is provided a formed chondrogenic or osteogenic (osteoconductive or osteoinductive) composition. In this aspect, demineralized bone or cartilage, mineralized bone or cartilage, or a paste containing mineralized or demineralized bone or cartilage is used as a vehicle for delivery of osteogenic or chondrogenic expressible nucleic acids. provide. In another aspect of the invention, methods for performing various surgical procedures using the compositions of the invention are described.

One object of the present invention is to provide a bone graft having a substantially natural mineral structure, reduced antigenicity (reduced immunogenicity), enhanced safety, and osteoinductive potential. That is.

Another object of the present invention is a cartilage transplant with a substantially natural mineral structure, reduced antigenicity (reduced immunogenicity), enhanced safety, and osteoinductive or chondrogenic potential. Is to provide a piece.

Another object of the present invention is to provide a spacer connecting the vertebrae that regenerates the intervertebral disc space to support the spinal column while promoting bone renewal and avoiding stress shielding.

Another object of the present invention is to use pins, suture anchors, interference screws, demineralized bone grafts (ligaments, oral maxillofacial plates, dowels, posterior approach lumbar interbody fusion implants (posterior). lumbar interbody fusion implant), traumatic screws and plates, pericardium (dura, plura, shoulder patch, and perioligament), wedges, tips and pastes (bone, cartilage, or other tissue). Nucleic acid encoding a growth factor (a group of factors including, but not limited to, a growth factor (bone morphogenetic protein, cartilage-derived morphogenetic protein, tissue growth factor (such as beta 1)) It is to be used in combination with.

One benefit of the present invention is that it solves many of the problems associated with bone and other implant material applications, regardless of the material of the allograft or xenograft. The process of antigen clearance described herein precludes immunogenicity or pathogenesis while preserving the natural microstructure of bone and other tissues described herein. This feature makes it possible to use allografts or xenografts, which are available in nearly unlimited supply. Enrichment of implants with nucleic acids encoding bone or cartilage growth factors or bone morphogenetic proteins allows them to become osteoinductive, thus eliminating the pain and associated risk of autologous graft retrieval .. Another benefit of the present invention is to provide a stable scaffold for bone, cartilage, or other tissue to renew during the fusion process or as new cartilage or tissue is produced.

Another object and advantage of the present invention is that the fast fusion rate allows the use of bone grafts without the need for metal cages or internal fixation.

Other objects and further advantages of the present invention will be apparent to those skilled in the art from the following description and the accompanying drawings.

[0031] To facilitate an understanding of the principles of the preferred embodiment of the described invention, with reference to the illustrated embodiment in the drawings will be described embodiments with reference to particular language. However, it is understood that this is not intended to limit the scope of the invention, and those of ordinary skill in the art to which the invention pertains will appreciate such modifications and further variations of the exemplified spacers, as well as the exemplified invention. It is intended to come up with such additional uses of the principle of.

The present invention provides bone and cartilage implant compositions, spacers, and surgical procedures. As a bone graft composition, an osteogenic substance (eg, bone morphogenetic protein (BMP
), A cartilage derived morphogenic protein, a growth factor, a bone or cartilage graft in combination with a nucleic acid encoding a peptide (eg, p15). Delivery of nucleic acids encoding the desired gene product results in uptake of such nucleic acids and expression of the encoded proteins, as is well known in the art. The nucleic acid can be so-called naked DNA or RNA, which contains suitable transcription and translation initiation and termination signals well known to those of skill in the art. The nucleic acid may also include a viral replication signal and may include a recombinant viral vector encoding the nucleic acid or gene desired for expression at a given transplant site.

The invention also includes, but is not limited to, bone, cartilage, or other tissues with reduced antigenicity, and growth factors (bone morphogenetic proteins, cartilage-derived morphogenetic proteins, tissue growth factors such as β1). ), A pin, a suture anchor, an interference screw, a demineralized or chlorinated bone graft (ligament, oro-maxillofacial plate, dowel, cervical vertebral posterior fixation implant, trauma) Screws and plates,
Provide pericardium (for dura, pullers, shoulder patches, and ligaments perimeter), wedges, tips, and pastes, including but not limited to. Thus, although the focus here is on bone grafts for spinal fusion, implants for a variety of orthopedic and non-orthopedic applications are based on this disclosure. It will be appreciated by those skilled in the art that it may be beneficial to treat B.sub.2 with an appropriate nucleic acid encoding a bone morphogenetic factor or a chondrogenic factor.

The method disclosed herein for a bone graft according to the invention to remove all of the cellular, fat, and non-collagenous proteins that otherwise bind to the bone graft composition. Can be processed according to. In a preferred embodiment, free collagen is also removed, leaving structural or bound collagen bound to the bone minerals that form the trabecular strut of bone. Such bone graft materials are largely devoid of non-collagenous proteins and unstructured collagen and depleted of fat, but still contain the natural crystalline structure of bone. Thus, such bone graft compositions of the present invention have the natural microstructure of bone without the risk of disease transmission or significant immunogenicity or antigenicity. The natural crystalline structure of bone is maintained by the presence of structural collagen bound to natural bone minerals. This results in a bone graft material with favorable physical and biological properties, including the ability to deliver nucleic acids, without associated immunogenicity or antigenicity.

The presence of structural collagen and the natural mineral structure of bone provides the same or nearly the same elasticity and radiopacity as bone. This material has sufficient elasticity and resilience to hold the molded body and yet is sufficiently rigid to maintain and secure the open space between the bone parts. Demineralized bone matrix treated with nucleic acid or autograft bone can also be used, but when xenograft bone is used it is preferred to remove non-collagenous non-structural proteins.

Bone or cartilage graft material of the present invention and BMP, CDMP (cartilage-derived morphogenetic protein)
When combined with nucleic acids encoding other growth factors, this composite material is an ideal bone graft substitute. This composite material has the natural calcium phosphate structure of bone. This facilitates the incorporation and replacement of the implant material and the absorption rate of the composite material of months is desirable. It is generally too fast, slow, or comparable to the unpredictable absorption rates of known materials. For example, allografts are generally absorbed within 12-16 months, whereas they may be absorbed too fast before fixation occurs due to an immunogenic reaction by the patient. .

The combination of nucleic acids encoding BMPs and other bone morphogenetic factors with the graft material according to the invention allows for autologous removal without the need for surgical intervention at secondary sites where pathological conditions may occur. The osteoinductive ability of the implant is obtained. The osteoinductive composite material of the present invention promotes bone growth and graft incorporation, resulting in faster fixation than fixation using only bone or cartilage graft material. Allografts alone generally require months to be incorporated, and may simply be wrapped in the patient's bone and not sufficient. The rapid fixation provided within about 5 months provided by the present invention compensates for the undesirable biomechanical properties of the implant and obviates the use of internal fixation and metal interbody fusion devices. The spacers of the present invention are not required to sustain a cyclic load of the spine for extended periods of time due to the high fixation rates that reduce the biomechanical requirements for the spacers. However, if desired, the composition of the present invention may be used in combination with an internal fixation device and optionally reinforced (see WO 98/56319, incorporated herein by reference). That).

If the bone graft material of the present invention has been treated to remove essentially all non-collagenous proteins and non-structural collagen, the graft will be an autograft,
It may be an allograft or a xenograft. Bone components that may cause disease or promote graft rejection of the patient's body are removed by the treatment processes disclosed herein. Heterologous bone (eg, bovine, ovine, porcine, canine, equine, or other bone) is available with virtually unlimited supply. Some bone morphogenetic factors are also available in unlimited supply thanks to recombinant DNA technology. Accordingly, the present invention solves all of the problems associated with autografts, allografts, and xenografts (supply, immunogenicity, disease transmission, and the need for secondary site surgical procedures). ing.

In the present invention, the bone mineral is a bone morphogenetic factor (eg, BMP, CDMP, peptide (eg, PMP).
15), and the discovery that it is an excellent carrier of nucleic acids encoding similar factors) provides further benefits. Hydroxyapatite, a mineral-like chemical composition in cortical bone, is an osteogenic factor-binding agent that controls the rate of delivery of certain proteins to anchorage sites. Calcium phosphate compositions such as hydroxyapatite are believed to bind to bone morphogenetic proteins and prevent BMP from prematurely escaping the spacer before fixation occurs. In addition, when the drug retains BMPs, the protein helps the mesenchymal stem cell bone within the device at a rate that aids in complete and rapid bone formation and ultimately fixation of the disc space. It is believed to cause conversion to producer cells or osteoblasts. The cartilage and bone graft-nucleic acid compositions of the present invention provide natural binding and controlled delivery of nucleic acids encoding bone morphogenetic proteins such as bone morphogenetic proteins while at the same time not inducing an unwanted immune response in the recipient. It has the benefit of having a bearing member consisting of.

The present invention also takes advantage of the discovery that metal-like cortical bone can be conveniently machined into the various shapes disclosed herein. In one aspect, the load bearing member is threaded on the outer surface. Machining surfaces (eg, threads) offer several benefits previously available only with metal implants. The use of threads can provide better spacer insertion control than smooth surfaces. This allows the surgeon to more accurately position the spacer, which is important for important neural and vascular structures of the spinal column.

Threads and the like also result in increased surface area, which facilitates the process of bone healing, gradual replacement to replace donor bone material, and fixation. These features also enhance post-operative spacer stability by engaging the adjacent vertebral endplates and anchoring the spacer to prevent extrusion. This is a major benefit over smooth grafts. The surface features also stabilize the bone-spacer interface, reducing micromotion and facilitating installation and fixation.
Methods for making such external bone features are disclosed in US Pat. No. 5,814,084, which is incorporated herein by reference.

The implant composition of the present invention can be prepared according to the methods disclosed herein. Human or animal sourced bone is obtained according to known procedures. Bone is washed to remove tissue and blood, then treated with drugs to remove cellular material, fat, non-collagenous proteins, and selectively to remove nonstructural collagen. Common agents include alcohols and peroxides. In a preferred embodiment, the bone material is also treated to remove free collagen leaving only bound or structural collagen bound to bone mineral. This reduces immunogenicity / antigenicity without compromising the structural integrity of the bone material. One preferred agent that removes free collagen and bound non-structural antigenic proteins as well as residual fat is a chaotropic agent (eg, urea, guanidinium hydrochloride, TritonX-
100, Tween, TNBP, etc.) in combination with alcohol and peroxide treatment. The bone material for the bone graft is then washed, preferably with sterile deionized water, and finally sterilized by a suitable method, including but not limited to gamma irradiation, gas phase peroxide treatment and the like.

Allograft, autograft, or xenograft bone nails or other appropriately shaped implants can be packaged fresh frozen or lyophilized (preferably lyophilized). it can. Sterilization can be accomplished by aseptic processing or by final sterilization with ETO, electron beam, or gamma irradiation (preferably gamma irradiation). Gamma irradiation makes it possible to obtain and process allografts or xenografts under conditions where the environment is not tightly controlled, as the final sterilization provides very high sterility.

The implants according to the invention are non-immunogenic, disease-free implant implants that remove all non-collagenous bone proteins and have a consistency that retains the natural mineral microcrystalline structure of bone and the desired morphology. It can be processed to leave strip material. The compositions of the present invention are preferred because they have the microstructure closest to natural bone of all known processed bone products. The bone product also has the radiopacity of natural bone and does not show the dark white image of the Spector and Geistlich bone product. The products of the invention also provide excellent absorbability, especially when used in combination with nucleic acids encoding bone morphogenetic proteins. Absorption has been found to beneficially occur within months, as opposed to the years required for Specter and Gastrich materials or the weeks of Urist products. When this material is used in combination with a nucleic acid encoding bone growth factor, this resorption time is sufficient to form the bone bridges required for fixation and bone healing.

The bone or cartilage material of the present invention is used in combination with an osteogenic composition or material containing a nucleic acid that expressably encodes a bone growth factor, protein, or peptide. The osteogenic nucleic acid can be added to the bone material by injecting a solution containing the osteogenic nucleic acid composition into the implant. Immerse the allograft, autograft, or xenograft for a time sufficient to allow the allograft, autograft, or xenograft to absorb the nucleic acid. Additional nucleic acid is added to the delivery vehicle that is placed around or within the allograft to add additional nucleic acid to the allograft, autograft, or xenograft made according to the methods of the invention. Can be used with. In some embodiments, the nucleic acid encoding the osteogenic composition can be packaged in a chamber formed within the body of material. Furthermore, the nucleic acid can be used in combination with a suitable protein growth factor. Various bone morphogenetic factors, growth factors, proteins, peptides, or nucleic acids can be applied to the interstitial space of bone or other cartilage implants by changing the pressure from high pressure to low pressure in a vacuum or pressure or in a suitable chamber or container. Can be tucked in. The composition may be added by the surgeon during surgery and the spacer may be provided with the previously added composition. In such cases, the osteogenic composition can be stabilized for shipping and storage, for example, by lyophilization. The stabilized composition can be rehydrated and / or reactivated with a sterile liquid (eg, saline or water) or body fluid added before or after implantation. Alternatively, a composition containing a lyophilized bone or cartilage matrix containing only expressible nucleic acid, or containing expressible nucleic acid and protein, peptide, or other growth factor, antibiotic, antitumor drug, anti-inflammatory drug, etc. It can be easily reconstructed in the object. The thus reconstituted implant may then be directly implanted into a suitable recipient or lyophilized with the added and absorbed osteogenic composition. In addition, the implants so treated can be treated or cultured with various cell types to obtain a cell-coated matrix that has incorporated expressible nucleic acids.

As used herein, the term “osteogenic composition” refers to virtually any substance that promotes bone growth or healing (such as natural, synthetic, and recombinant proteins, hormones, etc. (Including cells that express the factors, as well as nucleic acids that expressably express such factors). "Expressively coding" refers to a nucleic acid construct,
It is meant to include all signals required to achieve efficient expression of the encoded factor, including transcriptional promoters and terminators, enhancers and the like, which are well known to those of skill in the art. The osteogenic composition used in the present invention preferably expresses growth factor in an amount sufficient to stimulate or induce bone growth or healing of substantially pure osteoinductive factor (osteogenic protein). The nucleic acid encoding possible is contained in a pharmaceutically acceptable carrier. Preferred osteoinductive factors include, but are not limited to, recombinant human bone morphogenetic protein (rhBMP), CDMP, and nucleic acids encoding such factors. Most preferably, the nucleic acid is the bone morphogenetic protein rh
It encodes BMP-2, rhBMP-4, or a heterodimer thereof. The concentration of nucleic acid encoding rhBMP-2 or other growth factors (eg, TGF-β1, TGF-β2, p15, etc.) can be about 1 ng to 1 mg / g implant. Any bone, including BMP-1 to BMP-13, bone morphogenetic proteins termed CDMP1 and CDMP2, and various growth factors known in the art to be beneficial in inducing bone growth and tissue regeneration. Nucleic acids encoding forming proteins are contemplated. Nucleic acids encoding BMPs are well known in the art and are described in Wozney, incorporated herein by reference.
U.S. Pat. No. 5,187,076; Wozney et al. U.S. Pat. No. 5,366,875; Wang et al. U.S. Pat. No. 4,877,864; Wang et al.
, 922; U.S. Pat. No. 5,116,738 to Wang et al .; U.S. Pat. No. 5,0 to Wang et al.
U.S. Pat. No. 5,106,748 to Wozney et al .; and WO 93/00432 to Wozney et al .; WO 94/26 to Celeste et al.
893; and WO 94/26892 to Creste et al.

In addition, the growth factors disclosed in US Pat. No. 5,763,416, US Pat. No. 5,854,207
No. or No. 5,899,936, International Publication No. 97/38729, International Publication No. 95/2261.
All of the nucleic acids encoding the stimulatory factor disclosed in WO 99/06563 or in WO 99/06563 are also incorporated herein by reference for use in combination with the bone or cartilage matrix disclosed herein. Further, for example, U.S. Patent No. 5,635,374 (
Bone calci, disclosed herein by reference)
A nucleic acid containing a fication factor) so that it can be expressed can also be included in the graft composition of the present invention.

The choice of carrier material for the osteogenic nucleic acid composition is based on the desired application, biocompatibility, biodegradability, and interface properties. The composition that induces bone growth can be introduced into the pores of the bone material by any suitable method. For example, the composition can be injected into the pores of the implant. In other embodiments, the composition may be instilled onto the implant to stimulate osteoinduction, the implant may be dipped into a solution containing an effective amount of the composition, and the implant effective amount of the composition You may spray the solution containing a thing. Alternatively, the bone forming composition may be poured into the bone at elevated or reduced pressure or both. All of the methods of introducing proteins disclosed in US Pat. No. 5,854,207 are incorporated herein by reference as methods of introducing nucleic acids into bone or cartilage matrices.
In any case, the matrix nucleic acid osteogenic composition is completely immersed in the implant, the implant is coated and the matrix pores are exposed to the composition for a time sufficient to pour into the implant. A nucleic acid encoding an osteogenic factor, preferably BMP, is provided in lyophilized form and is a pharmaceutically acceptable liquid or gel carrier (eg, sterile water, saline, or any other suitable carrier). It can be dissolved and reconstituted. The carrier can be any suitable vehicle that can deliver the nucleic acid to the implant. Buffers are preferably added to this medium as is well known in the art. In one particular embodiment of the invention, the nucleic acid encoding rhBMP-2 is suspended or mixed in a carrier such as water, saline, MFR buffer, liquid collagen, or injectable dicalcium phosphate. . In the most preferred embodiment, the nucleic acid encoding BMP is added to the pores of the implant and then lyophilized. The nucleic acid graft-BMP composition can then be placed in a sterile container and stored at room or low temperature for storage and shipping. Alternatively, osteoinductive nucleic acid can be added during surgery.

Other osteoinductive protein carriers known in the art are available for delivering nucleic acids to chambers formed in bone or cartilage material, or to a location around a bone material implant site. Possible carriers include calcium sulphate, polylactic acid, polyanhydrides, collagen, calcium phosphate, polymeric acrylates,
Mention may be made of polyphoazine, polyamines, polycarbonates, etc., and demineralized bone. The carrier can be any suitable carrier capable of delivering the nucleic acid alone or the nucleic acid and the protein, peptide, or other biologically active agent. Most preferably, the carrier is capable of ultimate absorption by the body. One preferred carrier is the trade name Helistat®.
Absorbable Collage Hemostatic Agent
n Hemostatic Agent) at Integra Life Sciences Corporation (In
is an absorbable collagen sponge sold by tegra LifeSciences Corporation). Another preferred carrier is open cell polylactic acid polymer (OPLA). Other possible matrices for the composition are biodegradable, chemically defined calcium sulphates, calcium phosphates (eg tricalcium phosphate (TCP)).
And hydroxyapatite (HA), injectable dicalcium phosphate (BCP)
A)), as well as polyanhydrides. Other potential materials are biodegradable and of biological origin, such as bone or skin collagen. The additional matrix consists of pure proteins or extracellular matrix components. The osteoinductive material can also be a mixture of nucleic acid encoding BMP and a polymeric acrylate carrier (eg, polymethylmethacrylate, polyvinylacetate, polyhydroxyethylmethacrylate, etc.). To fill the chamber of the spacer of the invention, the carrier is preferably provided as a sponge that can be pressed into the chamber or as a strip or sheet that can be folded and fitted into the chamber. Preferably, the carrier has a width and length slightly greater than the width and length of the chamber. In a preferred embodiment, the carrier is dipped in the nucleic acid solution encoding rhBMP-2 and then pressed into the chamber. The sponge is held in the chamber by the compressive force of the sponge against the dowel wall surface. It may be desirable for the carrier to protrude from the chamber opening to facilitate contact between the osteogenic composition and the highly vascularized tissue surrounding the fixation site. The carrier can also be provided in several strips sized to fit inside the chamber. The strips can be placed against each other so as to occlude the interior. Similar to folded sheets, the strips can be aligned in several directions inside the spacer. Preferably, the osteogenic material, whether provided in a sponge, in a single folded sheet, or in several overlapping strips, has a length corresponding to the length and width of the chamber. Have.

A preferred carrier is a biphasic calcium phosphate ceramic. Hydroxyapatite / tricalcium phosphate ceramics are preferred due to the desired bioactivity and degradation rate in vivo. The preferred ratio of hydroxyapatite to tricalcium phosphate is about 0: 100 to about 65:35. Any size or shape of ceramic support that fits into the chamber formed in the load bearing member is contemplated. Ceramic block is Sofamor Danek Group BP4-62180 Ran
g-du-Fliers, France, and Bioland 132 Route d: Espagne, 3110
0 Commercially available from Toulouse, France. Of course, rectangles and other suitable shapes are contemplated. The osteoinductive factor is introduced into the carrier by any suitable method. For example,
The carrier can be dipped into a solution containing the factor.

The present invention also provides a spacer for maintaining the spacing between adjacent bones. The spacer comprises a body of bone or cartilage implant and a nucleic acid encoding bone growth factor. The bone source is any suitable (preferably vertebrate derived) bone material, including tibia, fibial, humerus, iliac and the like. The implant body of the present invention includes a flat spacer, a bone nail, a cortical ring, a bone tip,
And any other suitable shaped bone fragment. A preferred body is obtained from a long bone diaphysis having a medullary canal that forms a natural chamber in the implant.

In one particular embodiment shown in FIG. 1, the present invention provides a spacer 10 to maintain the spacing of adjacent bones in a patient. The spacer 10 comprises a load bearing member or body 11 sized and shaped to fit the spacing. Body 11 preferably comprises a natural bone or cartilage material that has been optimally treated to remove bound non-collagenous bone proteins. The bone material comprises natural collagen material and naturally associated bone mineral, but is substantially free of natural non-collagenous proteins. Alternatively, demineralized or partially demineralized bone may be used. The chemical composition of the bone material makes it possible to hold the shaped body elastically. The shape of the body is preferably molded and the body is machined to have the desired surface characteristics prior to treating the bone material according to the method of the present invention. However, in some embodiments, the bone mass is treated as disclosed herein and then shaped or machined to form a particular body.

Referring now to FIGS. 1 and 2, in one embodiment, the body 11 is molded as a dowel. A dowel-shaped body may be preferred when the bone to be fixed is a vertebra. The dowel 10 includes a wall 12 sized to engage within the intervertebral space (IVS) to maintain the IVS in proper physiologic orientation. The wall surface 12 forms an outer engagement surface 13 for contacting an adjacent vertebra. The wall 12 is preferably cylindrical, and the bone dowel 10 has a IVS height h between adjacent vertebra V, or the lowest lumbar vertebra L5 and sacrum as shown in FIG.
It has a width of diameter d that is greater than the height of the spacing of S.

In another embodiment shown in FIG. 3, the body is an interlocking nail 20 with a wall surface 22 having an engagement surface 23. A chamber 25 is formed through the wall surface 22. Preferably, the load bearing member is a bone graft obtained from a long bone diaphysis having a medullary canal forming a chamber 25. Such nails are sold by Regeneration Technologies Inc.
tion Technologies, Inc.) 1 Innovation Drive, Alachua, Florida 32615. The chamber 25 can be filled with an osteogenic composition containing nucleic acids encoding BMP, CDMP, etc. that stimulate osteoinduction. Preferably, a chamber 25 is formed through a pair of engaging outer surfaces 23 for maximum contact of the composition with the adjacent vertebral endplates. Referring now to FIG. 4, the spacer 20 is preferably a chamber 25
It comprises a solid protective wall 26 which can be arranged to protect the spinal cord from leakage or leakage of the material packed inside. In the front reaching method, the protective wall surface 26 is at the rear.
Preferably, the length of the osteogenic composition is longer than the length of the chamber (FIGS. 5 and 6),
When the spacer 20 is implanted between the vertebrae, the composition is placed inside the chamber 25 in contact with the endplates of adjacent vertebrae. This provides good contact between the composition and the endplates to stimulate osteoinduction.

Various features can be machined on the exterior surface of the dowels of the present invention. 1 shown in FIG.
In one aspect, dowel 20 comprises an engaging outer surface 23 forming a thread 24. again,
Referring to FIG. 1, in one aspect, a dowel 10 is provided that includes a tool engaging hole 19 in a wall surface 18 opposite a solid protective wall surface 16. Tool engaging holes 19 are provided on the surface of the dowel on the surgeon side and opposite the first thread 17. In the front reaching method, the tool engagement tool hole 19 is provided on the front surface of the dowel 10. Other machining features are contemplated on the outer surface or surface 23 that engages the bone. Such mechanical features include roughening such as knurling and ratcheting.

The spacers of the present invention can be inserted using conventional techniques and known tools. According to a further aspect of the invention, a method for implanting an interbody fusion spacer (eg, spacer 20) is contemplated. Sofamor Danek USA Laproscooic Bone Dowel Surgical Technique 1995,1800 Pyramid Place, Memphis, Tennessee 38132,1-
The spacers of the present invention can also be inserted using the laparoscopic technique described in 800-933-2635. The device of the present invention facilitates anterior interbody fusion using a much lower surgical approach than the open anterior retroperitoneal approach, which is standard for illness. It can be conveniently incorporated into a laparoscopic bone nail system. The system comprises templates, trephine, dilators, reamers, ports, and other equipment needed for laparoscopic dowel insertion.

The body may also comprise other shapes such as a cortical ring as shown in FIG. Such a cortical ring 50 is obtained by a transverse slice of the long bone diaphysis and comprises an upper surface 51 and a lower surface 52. The implant shown in FIG. 7 is adjacent to the upper surface 51 and the lower surface 52, and
And an outer surface 53 between the lower surface 52 and the lower surface 52. In one embodiment, the bone growth passage hole 53a,
Formed through the outer surface 53 to facilitate fixation. The holes 53a allow mesenchymal stem cells to enter the graft and allow bone growth proteins and nucleic acids encoding such proteins to dissipate from the graft. This may facilitate the incorporation of bone grafts and facilitate fixation by forming anterior and lateral bone bridges on and outside the device. In another aspect, the outer surface 53 defines a tool engagement hole 54 for receiving an implant tool. In a preferred embodiment, at least one of the upper surface 51 and / or the lower surface 52 is textured to securely grip the endplates of adjacent vertebrae. The textured surface may have teeth 56 on the ring 50 'as shown in FIG. 8 and waffle patterns 57 on the ring 50''as shown in FIG. If a cortical ring is used as the implant material, the ring 50 may be carved into a more uniform shape as shown in FIG. 7 or may be left in place as shown in FIG.

Implants were also used for the Smith-Robinson method for cervical fixation (
Smith, MD, GW and RA Robinson, MD, “The Treatment of Certain Cervical-Spine Di
sorders By Anterior Removal Of The Intervertebral Disc And Interbody Fus
ion) ” J. Bone And Joint Surgery 40-A 607-624 (1958), and Cloward, M.
.D, RB, "The Anterior Approa for the Removal of Ruptured Neck Discs (The Anterior Approa
ch For Removal Of Ruptured Cervical Disks) '', meeting of the Harvey Cush
ing Society, Washington, DC, April 22, 1958) and can be shaped into a square for convenient incorporation into currently used surgical procedures. In such procedures, the surgeon trims the endplates of the adjacent vertebral bodies to accommodate the implant after the disc has been removed. The end plates are generally arranged in parallel planes with high speed bars. The surgeon then generally scrapes the implant so that it fits tightly into the plane of the bone and retains the implant by compression between the vertebral bodies. The bone graft is intended to support the structure and promote bone ingrowth to secure the affected joint. The spacers of the present invention are provided with spacers of known size and dimensions,
This eliminates the need to scrape the implant. The present invention also eliminates the need for surgery on the donor, since the autograft osteoinductivity is obtained with allograft or xenograft grafts prepared according to the present invention. The spacer is used in combination with an osteoinductive material, including but not limited to nucleic acids that expressably encode growth factors that render the allograft or xenograft implant osteoinductive. Thus, the spacer of the present invention speeds patient recovery by shortening surgery time, avoiding painful donor surgery and inducing faster fixation. The following specific examples are provided to illustrate the invention and are not intended to limit the invention thereby.

Example 1 Removal of Antigen from Bone Graft Material The following procedure and modifications of its details are used to remove non-collagenous proteins from bone graft material. The bone graft material may be an allograft or a xenograft selected from cattle, pigs, horses, sheep, dogs and the like. This procedure removes proteins, fats, polysaccharides, glycosaminoglycans, and other non-collagenous antigens from the bone matrix, and can be performed in any order of steps, but in the order presented herein. Even if it carried out, the excellent result was always obtained. Performing this treatment is not essential when using autograft or allograft bone or cartilage material as the nucleic acid delivery matrix for the purposes of the present invention.

1. Peroxide treatment: Degrease bone tissue, remove blood and other proteins according to the following procedure,
Inactivates microorganisms that may be present in or on the bone. Prior to beginning this process, adhering adventitial tissue is removed from the bone. Bone tissue was placed in a container filled with a peroxide solution and immersed for about 15 minutes with agitation and / or sonication. b. The peroxide solution and the removed debris were discarded and the bone tissue was washed with warm sterile water.

At this stage, TNBP / Triton X-100 or similar solution (eg hydrogen peroxide / SDS
) Helps to remove additional nonstructural proteins and residual fat.

2. Acetone treatment: The residual adipose tissue was removed according to the following procedure: a. Bone tissue was placed in a container filled with acetone, heated to about 35 ° C to 40 ° C, and stirred for about 15 ° C. Soak for a minute. After cooling, repeat this step until fat disappears in solution (3-5 cycles are usually suitable). b. The bone is then washed with sterile water and dried.

Variations on this treatment can include using 99% isopropanol, hexane, and combinations of these solvents. Acetic acid or other acids (acetic acid, hydrochloric acid, hydrofluoric acid, phosphoric acid, citric acid, at this or different stages of the process
Treatment with formic acid, butyric acid, or other mixtures) is useful to create a slightly desalted, less antigenic bone graft, with concomitant graft strength, growth factor binding capacity. , Absorbs, removes acid-soluble proteins and loosely bound collagen, and further reduces antigenicity. We have from about 0% to about 25% or about 1% to about 10%, or normal bone mineral content, or
It has been discovered that reductions in mineral content as low as 1% to 5% provide significant benefits in reducing the antigenicity of bone compositions. The principle of the degree of desalting to be carried out is to remove as much mineral as possible while not reducing the compressive strength of the bone.
In order to achieve uniform, limited desalination, the graft is preferably contacted with an acid (eg, 1% acetic acid) for about 30 minutes (including the graft to ensure uniform acid permeation). Introduce acid into the vacuum chamber). When using an inorganic acid (eg, HCl), the acid strength or acid contact time should be small to avoid complete desalting of the implant. We found that limited removal of as little as 1-2% of normal bone mineral content resulted in even better predictability of strength of bone grafts treated in this way (shear stress measurement). Reduce the dispersion of values). Additional benefits of this treatment include lysis of acid-soluble proteins, efficient removal of SDS or other ionic solvents or contaminants, enhanced growth factor binding, reduced time to change the shape of the transplanted bone, and Further reduction in antigenicity is mentioned.

3. Urea treatment: Bound non-collagenous proteins were removed from bone tissue according to the following procedure. The tissue was transferred to a vessel sufficient to contain the tissue and an excess (about 5 times) urea solution (6M). b. Non-collagenous proteins were extracted from bone tissue with agitation for approximately 48 hours. c. The urea / protein solution was then discarded and the bone tissue was washed several times (about 3 times) with sterile water using at least 2 volumes of water. Each wash was continued with stirring for about 20 minutes. d. The final water wash was performed for 24 hours with agitation, after which the water was discarded and the tissue lyophilized.

The above procedure was performed using bovine bone blocks and cancellous chips. A 1 cm bone cube was cut from a bovine condyle. As a final sterilization step, the bone thus treated was lyophilized and then gamma irradiated. Bone graft material treated according to this procedure was implanted in a primate model. Little or no adverse immune reactions (swelling, inflammation) were detected. Further, the bone graft treated in this way was immersed in a bone morphogenetic protein and transplanted into a primate model. To the inside of the implant bovine bone,
And excellent induction of new bone growth in and around was detected without adverse immune reactions. Based on the success achieved by using a bone graft prepared as described herein to deliver a growth factor in the form of an active protein, express growth factor expressing cells or growth factor Successful delivery of nucleic acids encoding possible is expected. Produces growth factors (such as recombinant or natural) as disclosed in US Pat. No. 5,763,416, which is incorporated herein by reference or WO 99/06563, which is also incorporated herein by reference. ) Cell-injected, cell-coated, or nucleic acid construct-injected, or nucleic acid construct-coated allograft or xenograft bone allows bone growth without inducing an adverse immune response. Is expected to actively induce.

As an alternative to the above treatments, use of Guanidium Hydrochloride, TritonX-100, Tween, TNBP, etc., optionally including a combination of chaotropic agents and surfactants such as SDS (sodium dodecyl sulfate) Is mentioned. Examples of conditions for using these agents include 4M guanidinium hydrochloride and 1% TNBP / TritonX-10
It is possible to use 0.

Example 2 Preparation of diaphyseal cortical bone nails Consented donors (ie, donor cards or other forms of consent to serve as donors) were tested for various infectious diseases and pathogens (human immunodeficiency). Disease virus, cytomegalovirus, hepatitis B, hepatitis C, and several other pathogens). These tests can be performed by any of numerous means conventional in the art including, but not limited to, ELISA assays, PCR assays, or hemagglutination reactions. Such trials include (i) the American Association of Tissue Banks,
Tissue Bank Technical Manual, Technical Manual-Musculoskeletal Organization (Technical Manual fo
r Tissue Banking, Technical Manual-Musculoskeletal Tissues) M19-M20 pages;
(Ii) US Food and Drug Administration, Provisional Regulations, US Federal Government Gazette / Vol. 50, No. 238/1993 12
Tuesday, March 14 / Rules and Regulations / 65517, D. Infectious Disease Testing and Donor Screening
(Iii) MMWR / 43 / No. RR-8, Guidelines for Preventing Human Immunodeficiency Virus Transmission by Transplanting Human Tissues and Organs (Guidelines for Preventing Tr
ansmission of human Immunodeficiency Virus Through Transplantation of Hu
man Tissue and Organs) 4-7 pages; (iv) Florida Adminis
trative Weekly), Volume 10, Issue 34, August 21, 1992, 59A-1.OO1-O14 59A-l.005 (12)
(C), FAC, (12) (a)-(h), 59A-l.005 (15), FAC, (4) (a)-(8). In addition to a set of standard biochemical assays, whether the donor has been involved in a number of high-risk behaviors (eg, having multiple sexual partners, suffering from hemophilia, intravenous drugs). We interviewed the donor or his close relatives to see if they were involved in the use. After the donor was confirmed to be acceptable, the bone useful to obtain the nail was collected and washed.

Dowels were obtained from the long bone diaphysis as a transverse plug using a diamond blade bit, which was washed with water and cooled. Bits are commercially available (Starlite, Inc) and are generally circular with a gap inner diameter (intern of about 10 mm to about 20 mm).
al vacant diameter). The machine for obtaining intracortical dowels and cortical dowels consisted of a pneumatically operated small lathe made of stainless steel and anodized aluminum. It comprises a spring-loaded carriage that moves parallel to the cutter. The carriage rests on two runners, which are 1.0 inch stainless steel rods,
It has a travel distance of 8.0 inches. One runner has a set pin hole on the moving rod that prevents the carriage from moving when the set pin is placed in the desired hole. The carriage can be moved to the left or right using a knob with metric and English graduations. This allows the position of the implant to be determined. Above this carriage is a vise that cuts the dowel while holding the implant in place and holding it in place. The vise has a cutting part on the jaw that cleans the cutter. The lathe is equipped with a drive, which is an air motor with a valve controller that allows the desired RPM to be installed.

First, the carriage is pulled back by hand and locked in place with the set pin. Second, load the implant into a vise and align with the cutter. Third, start the machine and install the RPM by using the knob on the valve controller. Fourth, the set pin is used to load the implant onto the cutter and cut the dowel. When the cutter cuts to the end of the implant, the carriage stops on the set pin. Fifth,
Use sterile water to expel the dowel from the cutter. It can be fully autoclaved and is equipped with stainless steel vise and / or tightening fittings to hold the implant and cut the dowel. The implant can be positioned up to 0.001 "of 1 inch, which creates dowel uniformity during the cutting process.

The cutter used with the above machine can make dowel with diameter of 5 mm to 30 mm, and the size of the cutter is 10.6 mm; 11.0 mm; 12.0 mm; 13.0 mm; 14.0 mm; 16.0
mm; and 18.0 mm. The composition of the cutter is stainless steel with a diamond powder cutting surface that produces a very flat surface on the dowel wall. In addition, sterile water is used to cool the implant and / or dowel and remove debris from the implant and / or dowel at the same time the dowel is cut (water injection). Water falls down through the center of the cutter to irrigate and wash the dowel under pressure. In addition, the water helps release the dowel from the cutter.

Next, the bone marrow is removed from the medullary cavity of the dowel and the medullary cavity is washed to create a chamber. The interior of the chamber may be scraped or machined as desired, allograft,
It may be occluded with any desired osteogenic material, including autografts, ceramics, growth factors and the like. The final machined product may be stored, frozen, or lyophilized and vacuum packed for later use. In one embodiment of the invention, dowels are made with a nucleic acid encoding BMP. In another embodiment, dowels are made with a mixture of nucleic acids encoding a variety of different growth factors.

Example 3 Threaded Dowel Cortical Osteocortical Dowels are prepared as described above. The plug is then preferably machined in a Class 10 clean room to the desired dimensions. The machining can preferably be carried out on a lathe (eg a jewelery lathe) or the machining tool can be specially designed to suit this purpose. Then, make a hole in the front wall surface of the dowel. An internal thread is then cut into this hole to accommodate the threaded insertion tool. The thus-prepared dowel is treated with nucleic acid as described in Example 2.

Example 4 Preparation of bone nail -rhBMP-2 nucleic acid composite material by dripping A threaded bone nail is obtained by the method described above. Lyophilized rh as follows
A vial containing 4.0 mg of nucleic acid encoding BMP-2 was made up with 1 mL of sterile water for injection (Abbott Laboratories) to give a 4.0 mg / mL solution.
Using a 1.3cc syringe and a 22G needle, 1.0 mL of sterile water for injection was lyophilized.
Slowly inject into vial containing nucleic acid encoding hBMP-2. 2. Gently swirl the vial until a clear liquid is obtained. Do not shake.

The appropriate rhBMP-2 nucleic acid concentration is obtained according to the following dilution scheme. This dilution gives a sufficient amount for two dowels. Dilution is performed as follows. Using a 1.5cc syringe, transfer 4.0 mL of MFR906 buffer (Genetics Institute) to a sterile vial. Using a 2.1 cc syringe, transfer 0.70 mL of reconstituted rhBMP-2 nucleic acid into a vial containing buffer. 3. Gently swirl to mix.

Using a 1.3 cc syringe and a 22G needle, 2.0 mL of 0.60 mg / mL rhBMP2 nucleic acid solution is slowly dropped into the bone nail. 2. Immediately transplant.

As an alternative to the above, the bone graft is lyophilized and then contacted with a solution containing nucleic acids encoding bone morphogenetic proteins, growth factors to reconstitute the lyophilized bone nail. In the reconstitution process, the dowels draw nucleic acids encoding growth factors or other osteogenic compositions (natural or recombinant) into the interstices of the bone matrix. The composition of nucleic acids may also include proteins, peptides, or other biologically active components.

Example 5 Preparation of Allograft Bone or Xenograft Bone-BMP Nucleic Acid Composite by Immersion 1. Lyophilized nucleic acid encoding rhBMP-2 was treated with sterile water for injection as in Example 4. Reconstruct. 2. Transfer the sterile allograft or xenograft bony nails to a sterile "dip" container. 3. Add the reconstituted nucleic acid encoding rhBMP-2 to the soaking solution so that the allograft is completely immersed in the solution. 4. Allograft or xenograft bone nails are placed in a solution of rhBMP-2 encoding nucleic acid in an amount of 30-60 to allow the graft to absorb nucleic acids and any other components added to the solution.
Soak for a minute.

To increase the efficiency of loading nucleic acids encoding BMPs or other growth factors, about 1
The autograft, allograft, or xenograft is contacted with a nucleic acid encoding such a factor under vacuum while swirling for 5 minutes.

Example 6 Bone-Nail Filled with Nucleic Acid / Collagen Composition Encoding BMP-2 The method of Examples 1-5 provides threaded nails. A vial containing 4.0 mg of lyophilized nucleic acid encoding rhBMP-2 is made up with 1 mL of sterile water for injection or saline solution to give a 4.0 mg / mL solution as follows. Using a 1.3 cc syringe and 22 G needle, 1.0 mL of sterile water for injection or saline solution is slowly injected into the vial containing the lyophilized nucleic acid encoding rhBMP-2. 2. Gently swirl the vial until a clear solution is obtained. Do not shake. Obtain the appropriate nucleic acid concentration according to the following dilution scheme. Dilute as follows.

Using a 1.3 cc syringe, transfer 2.5 mL of MFR-842 buffer (Genetics Institute) to a sterile vial. Using a 2.1 cc syringe, transfer 0.30 mL of 4.0 mg / mL reconstituted rhBMP-2 encoding nucleic acid to a vial containing buffer. 3. Gently swirl to mix.

The rhBMP-2 nucleic acid solution was added to the helistat sponge (Genetics Institute) as follows. 1. Using sterile tweezers and scissors, cut a 7.5 cm x 2.0 cm helistat piece from a 7.5 x 10 cm (3 inch x 4 inch) sponge. Using a 2.1 cc syringe and a 22-G needle, approximately 0.8 mL of 0.43 mg / mL rhBMP-2 nucleic acid solution is slowly and evenly dropped on the helistat sheet. 3. Using sterile tweezers, loosely fill the sponge chamber with the sponge. The remaining 0.8 mL of 0.43 mg / mL rhBMP-2 nucleic acid is injected through the chamber opening into the sponge in the dowel using a 4.1 cc syringe and a 22-G needle. 5. Immediately transplant.

Example 7 Dowel Nail Packed with Nucleic Acid / HA / TCP Composition Encoding rhBMP-2 By the method of Examples 1-5, threaded dowel is obtained. A vial containing 4.0 mg of lyophilized nucleic acid encoding rhBMP-2 is made up with 1 mL of sterile water for injection or saline solution to give a 4.0 mg / mL solution as follows: 1.3 cc syringe and 22 G needle. Using 1.0 mL of sterile water for injection, lyophilized rhBMP-2
Slowly pour into vial containing nucleic acid. 2. Gently swirl the vial until a clear solution is obtained. Do not shake. 2
Wet a cylindrical block of phase hydroxyapatite / tricalcium phosphate (BioRad) with 0.4 mg / mL rhBMP-2 nucleic acid solution. The nucleic acid-ceramic block is packed into a dowel chamber and then the dowel thus packed is implanted.

Example 8 Allograft or Xenograft Bone Chip-Preparation of Nucleic Acid Composite Material 1. Allograft or xenograft bone chips were collected and processed according to Example 1.
Prepare to make an allograft or xenograft bone chip. 2. Lyophilized nucleic acid encoding rhBMP-2 is reconstituted with sterile water for injection or saline as described in Example 4. 3. Transfer the sterile allograft or xenograft bone chips to a sterile "dipping" container. Preferably, the bone chips are reconstituted upon contact with the BMP nucleic acid solution, which results in BMP
The bone chips are first lyophilized so that the nucleic acid solution is absorbed in the chip gap. 4. The allograft or xenograft was reconstituted to be completely immersed in the solution
Place the rhBMP-2 nucleic acid in a dip container. 5. Immerse allograft or xenograft bone chips in rhBMP-2 nucleic acid solution for 30-60 minutes. 6. Using sterile forceps, remove the allograft or xenograft bone tip from the dip container and into the posterolateral groove at the level of the spine to be fixed, or any other where bone fixation or repair is desired. Place on any bone site.

Injecting gelatin into bone chips prepared as described above using the following procedure,
The gelatin nucleic acid sponge can then be made by freeze-drying this composition. Similar to Example 5, when a nucleic acid encoding BMP or other growth factor is contacted with the implant under vacuum, the efficiency of loading such nucleic acid is increased.

Example 9 Cortical Rings-Preparation of Composite Materials Cortical rings are obtained as transverse slices of the human long bone diaphysis. Alternatively, cortical rings are prepared using the method described in Example 1 to create a reduced antigenicity cortical ring. The cortical ring is made into a hollow square ring. As described in the previous example, the ring is filled with an osteogenic composition containing nucleic acid that expressively expresses a growth factor.

Example 10 Spacer A D-type cervical spacer is obtained as a transverse slice of the diaphyseal of the long bone and processed according to the method of Example 1. The outer surface of the wall is formed by machining the slice into a D shape. Knurling with a standard milling machine provides the spacer engagement surface. Then, a hole is made in the front wall surface of the spacer. An internal thread is then cut into this hole for engagement with the threaded insertion tool. The spacer chamber is then loaded with the osteogenic nucleic acid composition as described in the previous examples.

Example 11 Anterior interbody cervical fusion Access to the cervical spine from the anterior according to known surgical procedures. A composite material of the present invention containing a nucleic acid encoding an osteogenic factor is placed in the interdisc space.

Example 12 Posterior Lateral Fixation The spine is reached posterolaterally according to known surgical procedures. A composite material of the invention containing a nucleic acid encoding a bone morphogenetic protein is placed between adjacent vertebrae.

Example 13 Combined Use of Composite Material and Binding Matrix Nucleic acid-poured engineered allograft or xenograft tips were added to the binding matrix to keep the tips apart. Improve its handling. The chips are added to gelatin and water to form a paste or slurry and then optionally lyophilized into sheets or any other desired form. During surgery, a surgeon hydrates a gelatin, implant, nucleic acid composite with a solution, optionally containing a nucleic acid encoding an osteoinductive factor. Alternatively, during manufacture, the nucleic acid solution can be lyophilized on a hemostatic sponge or other biologically acceptable sponge. Alternative binding matrix materials include gelatin, glycosaminoglycans, hyaluronic acid, polymers, proteins, and other suitable materials. With or without added demineralized bone matrix
The compositions described herein have applications in various fields of orthopedics. For example, an appropriately balanced amount of demineralized bone, gelatin, growth factors, etc. may be used to prepare the preformed shape. Gelatin is present in sufficiently high concentration that it is semi-solid, malleable solid, or viscous at temperatures above the recipient's normal body temperature, but normal when transplanted to the recipient. Compositions that gel or solid at body temperature are highly desirable. For such applications, about 1% to 2% depending on the average molecular weight of the gelatin used in such compositions.
A gelatin concentration of 5% is generally sufficient. In addition, gelatin is present in sufficiently high concentrations that it is solid above the recipient's normal body temperature, but is malleable at a temperature just above normal body temperature, and as a result, is substantially solid at higher temperatures. It is also highly desirable to have a composition that can be made into a solid of any desired shape, such that the composition retains its molded shape when implanted in a recipient. Generally, a gelatin concentration of about 10-40%, depending on the molecular weight of the gelatin used in such compositions, is sufficient for this purpose. Nucleic acid encoding a growth factor can be added to the binding matrix.

Example 14 Characterization and Uses of Bone and Cartilage Matrices Containing Nucleic Acid Bone or cartilage implants in combination with bone growth factor-encoding nucleic acids provide superior results compared to other known implant materials. can get. The faster the fixing speed, the more quickly the mechanical strength increases. The graft of the present invention is an excellent nucleic acid carrier for controlled release of nucleic acid encoding BMP or other bone morphogenetic composition (growth factor, cartilage-derived morphogenetic protein, nucleic acid encoding BMP or other growth factor) to a fixed site. Is. Structural The presence of collagen and the natural mineral structure of bone provides the same or nearly the same elasticity and radiopacity as bone. This material has sufficient resilience and elasticity to hold the formed body, and yet is sufficiently rigid to maintain and secure open spaces between bone parts.

Example 15 Cartilage and Other Tissues as Nucleic Acid Delivery Matrix In a manner similar to that used to prepare nucleic acid-injected implants, cartilage implants, reduced antigenicity cartilage implants, and Other tissues can be made by treating such tissues with nucleic acids encoding any desired factor, including but not limited to osteogenic factors, pro-angiogenic factors, and the like. As described above for bone, the antigenicity of such tissues can be reduced by treatment with an appropriate chaotropic agent. The cartilage and other tissues so treated are then contacted with a variety of growth factor-encoding nucleic acids, cells, proteins, antifungal agents, antibiotics, antitumor agents, analgesics, etc., as described above. Let

Although the present invention has been illustrated and described in detail in the drawings and foregoing description, the drawings and the foregoing description are to be considered as illustrative and not limiting in character.
It will be appreciated that only the preferred and best embodiments have been shown and described, and it is desired to protect all changes and modifications that are within the scope of the invention.

[Brief description of drawings]

FIG. 1 is a top perspective view of a bone dowel implant according to US Pat. No. 5,814,084 treated according to the method of the present invention to include nucleic acid encoding a growth factor.

FIG. 2 shows placement of a bilateral dowel between L5 and the sacrum using the bone dowel shown in FIG. 1 containing a nucleic acid encoding a bone growth factor.

FIG. 3 is a perspective view of the cortical bone dowel shown in FIG. 1 having the appearance characteristics of a chamber and threading, and containing a nucleic acid encoding an osteogenic factor.

FIG. 4 is a perspective view of a bone dowel according to the present invention viewed from the side.

FIG. 5 is a sectional view of a bone dowel according to the present invention.

FIG. 6 is a side elevational view of the bone dowel shown in FIG.

FIG. 7 is a cortical annulus packed with osteogenic material containing nucleic acid encoding an osteogenic factor.

FIG. 8 illustrates one embodiment of the cortical bone rings provided by the present invention.

FIG. 9 is another embodiment of a cortical ring provided by the present invention.

[Procedure for Amendment] Submission for translation of Article 34 Amendment of Patent Cooperation Treaty

[Submission date] August 21, 2001 (2001.8.21)

[Procedure Amendment 1]

[Document name to be amended] Statement

[Name of item to be amended] Claims

[Correction method] Change

[Contents of correction]

[Claims]

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Claims (51)

[Claims] 1. An allograft, autograft or xenograft composition comprising a nucleic acid or a mixture of nucleic acids encoding one or more tissue regenerating, osteogenic or chondrogenic growth factors. 2. The composition of claim 1, wherein the allograft, autograft, or xenograft composition is bone tissue or cartilage tissue. 3. The implant is engineered to remove substantially all associated non-collagenous or non-structural collagen proteins and the material comprises a natural collagen material. 2. The composition of the bone or cartilage graft according to 2. 4. The composition of the bone or cartilage implant of claim 2, wherein the implant is desalinated or partially desalinated. 5. The bone or cartilage implant composition of claim 2, wherein the implant is not desalted. 6. A step of removing bonded non-osseous foreign material from a bone graft to provide a cleaned bone graft, for providing a cleaned defatted bone graft. Prepared by a process comprising the steps of contacting the cleaned bone graft with a degreasing solution, and contacting the cleaned defatted bone graft with a chaotropic agent to remove non-collagenous or nonstructural collagen proteins. The composition of the bone or cartilage graft according to claim 3. 7. The chaotropic agent is urea, guanidinium hydrochloride, Tween, T
The composition of the bone or cartilage implant according to claim 6, which is selected from riton X-100 and a mixture of these agents. 8. The bone or cartilage implant composition of claim 2, wherein the nucleic acid encoding the bone morphogenetic gene product incorporated into the implant comprises an amount effective to stimulate bone growth. 9. The bone or cartilage implant composition of claim 2, wherein the bone or cartilage is human, bovine, ovine, equine, porcine, or canine bone, or a combination thereof. 10. A spacer, pin, suture anchor, alone or in combination with a nucleic acid encoding a growth factor (including but not limited to bone morphogenetic proteins, cartilage-derived morphogenetic proteins, tissue growth factors (β1, etc.)), Interference screw, demineralized bone graft (including ligament weakened bone, cartilage, or other tissue, ligaments, oral maxilofacial, dowel, lumbar posterior fusion implant Includes posterior lumbar interbody fusion implants, traumatic screws and plates, dura, plura, shoulder patches, and pericardium for perioligament, wedges, tips and pastes, The composition of a bone or cartilage implant according to claim 2, which has been machined to form (but is not limited to) these. 11. The bone or cartilage implant of claim 10, for maintaining a space between a pair of adjacent vertebrae in the spine, including a body having a size and shape that fits within the disc space. Composition. 12. Cleaning, wherein the bone graft is either shaped to form spacers prior to cleaning, degreasing and contacting, or after cleaning, degreasing and contacting. Removing bonded non-osseous exogenous material from the bone graft to provide a cleaned bone graft, cleaned bone graft to provide a cleaned defatted bone graft 12. A method comprising: contacting a defatted solution with a delipidating solution; and contacting a defatted bone graft that has been cleaned to remove non-collagenous or unstructured collagen proteins with a chaotropic agent. Of a bone or cartilage implant of. 13. An implant having an upper wall for contacting an upper vertebra, a lower wall for contacting a lower vertebra, and a lateral wall adjacent to and between the upper and lower walls and defining a through hole. 13. The composition of the bone or cartilage implant of claim 12, which is defined. 14. The composition of the bone or cartilage implant of claim 13, wherein the implant is derived from a femoral ring. 15. The bone or cartilage implant composition of claim 13, wherein the implant is derived from a bone nail. 16. The bone of claim 13, wherein the wall defines a chamber, the chamber being filled with a pharmaceutically acceptable carrier having a nucleic acid encoding a bone growth factor dispersed therein. A composition of cartilage implants. 17. The composition of a bone or cartilage implant according to claim 2, wherein after implantation, the implant is substantially radiopaque for the bones of the vertebrae between which the spacer is inserted. 18. A bone mineral having a natural crystalline structure of bone and a native collagen material, and at least one bone morphogenetic protein encoded within the material in an amount effective to stimulate bone growth. A composition comprising bone material processed with the nucleic acid according to claim 1. 19. A bone growth-stimulating effective amount of a nucleic acid encoding essentially at least one bone morphogenetic protein, which essentially comprises natural forms of structural bone collagen and natural bone mineral. An elastic body that is substantially free of non-collagenous proteins and non-structural collagen proteins. 20. A surgical method for stabilizing the spine, comprising the steps of: exposing a portion of each adjacent vertebra in need of stabilization; and wherein the material is at least one osteogenic factor. Placing a processed bone material, including a bone matrix, infused or coated with a nucleic acid that encodes the following: in a region between portions of adjacent vertebrae. 21. The surgical method of claim 20, wherein the bone material is formed as an elastic body defining a chamber within which the osteogenic composition is filled. 22. The surgical method of claim 21, wherein the bone morphogenetic protein is dispersed in a pharmaceutically acceptable carrier within the bone material. 23. The method of claim 20, wherein a portion of the spinal column is on the posterolateral surface of the spine. 24. The method of claim 20, wherein the material comprises chips. 25. The method of claim 20, wherein the nucleic acid encodes a bone morphogenetic protein, cartilage-derived growth factor, tissue growth factor, bone mineralization factor, or a complex thereof. 26. A composition comprising a chip or a soft chip and a nucleic acid encoding a bone morphogenetic protein, a cartilage-derived growth factor, a tissue growth factor, a bone mineralizing factor, or a complex thereof, which comprises a protein, a peptide, A composition optionally comprising an anti-tumor drug, an anti-inflammatory drug, an antibiotic, or a complex thereof. 27. The composition of claim 26, further comprising a binding matrix and chips disposed within the matrix. 28. The composition of claim 27, wherein the matrix comprises gelatin. 29. A semi-liquid, malleable solid or viscous liquid above the recipient's normal body temperature, but at a high enough concentration to become a normal body temperature gel or solid when transplanted into the recipient. 29. The composition of claim 28, which comprises gelatin. 30. A composition that is solid at temperatures above the normal body temperature of the recipient, but which is substantially free of any desired shape when the composition is made at an elevated temperature and implanted into the recipient, where the composition retains its shape. 29. The composition of claim 28, comprising a high concentration of gelatin sufficient to be a malleable solid at such moderately elevated temperatures. 31. A composition of bone graft, which is contacted with acid prior to injecting nucleic acid into the graft composition. 32. The bone graft is acetic acid, hydrochloric acid, hydrofluoric acid, phosphoric acid, citric acid, formic acid, butyric acid, or a mixture thereof, and the bone graft usually has a bone mineral content of about 0% to 2%.
32. The composition of the bone graft according to claim 31, which is contacted so as to be desalted to about 5%. 33. The bone graft composition of claim 32, wherein the bone graft is normally desalinated to the extent of about 1% to 10% of bone mineral content. 34. The bone graft of claim 33, wherein the bone graft is normally desalinated to the extent of about 1% to about 5% of the bone mineral content. 35. A method of making a tissue graft for transplantation into a recipient in need thereof comprising the steps of: (a) cleaning a tissue piece of unwanted material; (b) tissue. Contacting the thus cleaned tissue section, in which the nucleic acid encodes a product desired to be expressed in the recipient of the implant, with the nucleic acid under conditions sufficient to achieve absorption or adsorption of the nucleic acid; and ( c) contacting the nucleic acid contacted with the tissue section with a chemical or energetic agent that sufficiently removes or inactivates the microorganisms but does not inactivate the nucleic acid. 36. The method of claim 35, wherein the tissue is bone. 37. The method of claim 35, wherein the bone is lyophilized and then reconstituted in a solution containing nucleic acids. 38. The method of claim 35, wherein the bone is immersed in a solution containing nucleic acid. 39. Mineral content reduced to about 0% to 25% of normal bone mineral content at any stage prior to contacting the bone with nucleic acid and further by contacting the bone with sufficient acid for a sufficient time. 37. The method of claim 36, wherein the method produces bone with reduced antigenicity. 40. The implant further comprising a biologically active agent selected from the group consisting of growth factors, antibiotics, antitumor agents, antifungal agents, antiviral agents, and combinations thereof. 40. The method of claim 39, wherein the biologically active agent is contacted under conditions that allow sufficient uptake into the reduced antigenic bone matrix. 41. An allograft, autograft, or xenograft composition comprising a nucleic acid or mixture of nucleic acids encoding one or more tissue-regenerating, osteogenic or chondrogenic growth factors. 42. An allograft, autograft, or xenograft composition comprising bone, cartilage, fascia lata, tendon, ligament, peritoneum, dura, pericardium, muscle, vasculature, epidermis, dermis. ,
The composition of claim 1, which is or a combination thereof. 43. The composition of claim 41, wherein the composition is in the form of a patch. 44. The growth factor is epidermal growth factor (EGF) or transforming growth factor α.
(TGF-α), transforming growth factor β (TGF-β), human endothelial cell growth factor (ECGF),
Granulocyte macrophage colony-stimulating factor (GM-CSF), bone morphogenetic protein (BMP)
, Nerve growth factor (NGF), vascular endothelial growth factor (VEGF), fibroblast growth factor (FGF
42), insulin-like growth factor (IGF), platelet-derived growth factor (PDGF), cartilage-derived morphogenic protein (CDMP), or a combination thereof. 45. The composition of claim 42, wherein the composition is dermal tissue. 46. The composition of claim 44, wherein the growth factor is VEGF, TGF, ECGF, FGF, or a combination thereof. 47. Repairing damaged tissue, comprising obtaining a piece of tissue infused with a nucleic acid or mixture of nucleic acids encoding one or more growth factors; and implanting the piece of tissue in a patient in need thereof. Or a method of stimulating tissue production. 48. The method of claim 47, wherein the tissue is vascular tissue and the implanting comprises connecting the piece of tissue to an artery or vein. 49. The method of claim 47, wherein the one or more growth factors have vascular tissue producing properties. 50. The method of claim 48, wherein the one or more growth factors is VEGF, TGF, ECGF, FGF, or a combination thereof. 51. The method of claim 47, wherein the tissue is in the form of a patch and the patch is attached to the heart of a patient in need thereof.