Journal of the American Academy of Orthopaedic Surgeons 6 Advances in molecular biology, cell biology, and polymer chemistry are providing novel approaches for treating musculoskeletal disorders. These advances are facilitating bio- logic approaches that complement and could even replace traditional biomechanical techniques in ortho- paedic surgery. The explosion of research has resulted in a myriad of potential applications for these innovative biologic approaches. Many probably will never reach clinical fruition; however, effective implementation of only a few may revolutionize the way we manage certain musculoskeletal disorders. Clearly, orthopaedic surgeons must have a fundamental understanding of the terminology (Table 1) and techniques of these biologic ad- vances, and of current research, to be able to interpret the literature, direct future investigations, and best serve their patients. Tissue Engineering Tissue engineering is the science of creating living tissue to replace, repair, or augment diseased tissue. 1 Tissue engineering thus refers to a broad variety of techniques. The engineered tissue is created either entirely in vivo or, in an ex vivo technique, is created in vitro and subsequently implanted into the patient. Regardless of the tech- nique, however, tissue engineering requires three components: a growth- inducing stimulus (growth factor), responsive cells, and a scaffold to support tissue formation. Growth Factors and Gene Therapy Cytokines are small, soluble pro- teins that influence cell behavior. A subset of cytokines, known as growth factors, promotes cell mito- sis. The term growth factor, how- ever, often is used generically in reference to any number of cyto- kines that promote cell division, maturation, and/or differentiation. Recent advances in molecular biology have facilitated the genetic sequencing of various cytokines. Consequently, the genetic sequences can be inserted into cells of labora- tory animals, producing large quan- tities of “recombinant” human cyto- kines. A particular cytokine often has multiple, diverse effects, and these can vary depending on the different cell types. Determining the influence and pharmacokinetics of various cyto- kines is an area of active, ongoing investigation. In vitro research typ- ically is performed to identify the optimal cytokine for a particular Dr. Musgrave is Chief Resident, Department of Orthopaedic Surgery, University of Pittsburgh Medical Center, Pittsburgh, Pa. Dr. Fu is David Silver Professor and Chairman, Department of Orthopaedic Surgery, University of Pittsburgh Medical Center, Pittsburgh. Dr. Huard is Associate Professor, Departments of Orthopaedic Surgery, Molecular Genetics and Biochemistry, and Bioengineering, University of Pittsburgh, Pittsburgh. Reprint requests: Dr. Huard, Room 4151, Rangos Research Center, 3705 Fifth Avenue, Pittsburgh, PA 15213-2583. Copyright 2002 by the American Academy of Orthopaedic Surgeons. Abstract A new biologic era of orthopaedic surgery has been initiated by basic scientific advances that have resulted in the development of gene therapy and tissue engi- neering approaches for treating musculoskeletal disorders. The terminology, fundamental concepts, and current research in this burgeoning field must be understood by practicing orthopaedic surgeons. Different gene therapy approaches, multiple gene vectors, a multitude of cytokines, a growing list of potential scaffolds, and putative stem cells are being studied. Gene therapy and tissue engineering applications for bone healing, articular disorders, interverte- bral disk pathology, and skeletal muscle injuries are being explored. Innovative methodologies that ensure patient safety can potentially lead to many new treat- ment strategies for musculoskeletal conditions. J Am Acad Orthop Surg 2002;10:6-15 Gene Therapy and Tissue Engineering in Orthopaedic Surgery Douglas S. Musgrave, MD, Freddie H. Fu, MD, and Johnny Huard, PhD Perspectives on Modern Orthopaedics Douglas S. Musgrave, MD, et al Vol 10, No 1, January/February 2002 7 application. The optimal method by which to deliver the cytokine in vivo also must be determined. The simplest method is to inject or im- plant the recombinant protein di- rectly into the anatomic region of interest. However, the inherently short half-lives of recombinant pro- teins and the dilutive effects of body fluids limit this technique. Consequently, the injected cyto- kines may not be present in ade- quate concentrations and at the critical time periods to effect a maximal response. Therefore, in- vestigators have devised two basic approaches to deliver temporary, sustained quantities of a desired protein. The first method is to impreg- nate the cytokine onto a polymer scaffold. Such a method requires a thorough understanding of the chemical interactions between the protein and the scaffold, the bind- ing characteristics of the protein to the scaffold, and the pharmacoki- netics of both the protein and the scaffold. The second method is to deliver the gene encoding for the protein into the patient, resulting in in vivo cytokine production. When the gene is delivered only to a spe- cific region of the body and not systemically, the method is termed regional gene therapy. Gene therapy is simply the transfer of a particular gene into a cell so that the cell transcribes the gene into messenger ribonucleic acid (mRNA); the cell’s ribosomes then translate the mRNA into a protein (cytokine). To accomplish this, the particular gene must be packaged and inserted into the Table 1 Common Terms Used in Gene Therapy Term Definition cDNA Complementary DNA. DNA created from a protein’s mRNA by enzyme reverse transcriptase. Contains only exons. mRNA RNA that is produced by transcription of DNA. This genetic material leaves the nucleus, moves to the cytoplasm, and is translated by ribosomes into a protein. RNA A string of ribonucleotides that is similar in structure to DNA. There are several classes of RNA, including messenger RNA (mRNA), transfer RNA (tRNA), and ribosomal RNA (rRNA). Nucleotide Building block of DNA and RNA comprising a base, a deoxyribose or ribose sugar, and a phosphate. Exon A piece of DNA encoding a gene that is transcribed into mRNA and then translated into a protein (that is, a nucleotide sequence of a gene that encodes for a specific protein). Intron A piece of DNA of unknown function that is interspersed between exons (that is, a noncoding nucleotide sequence of a gene). Transcription The process of conversion of DNA into RNA (that is, creation of an mRNA from DNA). Transduction Gene transfer using viral vectors. Transfection Gene transfer using nonviral vectors. Translation The process of conversion of mRNA into an amino acid sequence (that is, creation of a protein from mRNA by ribosomes). Plasmid A self-replicating piece of DNA naturally found in bacteria and yeast. Plasmids are commonly used to carry foreign genes into cells for the production of recombinant DNA pharmaceuticals and in gene expression studies. Liposome A gene-delivery system based on the production of lipid bodies that contain pieces of DNA. These lipid-DNA complexes fuse with the cell surface and are internalized by phagocytosis, depositing the encapsulated DNA into the target cells. Promoter A regulatory element in DNA that functions to induce transcription of a gene. Cytokines Small proteins that influence cell behavior. Ex vivo/In vitro gene transfer Genetic manipulation of cells outside the host (body). In vivo gene transfer Genetic manipulation of cells within the host (body). Vector A disabled virus or DNA structure used as a vehicle to transfer genes into cells. Gene Therapy and Tissue Engineering Journal of the American Academy of Orthopaedic Surgeons 8 desired cells. The protein’s mRNA is reverse-transcribed in the labora- tory by the enzyme reverse trans- criptase to create a complementary deoxyribonucleic acid (cDNA). Because the cDNA is created di- rectly from mRNA, the noncoding nucleotide sequences (introns) of the gene are not present, leaving only the protein-coding nucleotide sequences (exons). This fact distin- guishes a protein’s cDNA from its DNA sequence found naturally, which includes both introns and exons. The cDNA is inserted into a cir- cular plasmid with various non– amino acid-coding sequences at its leading and trailing ends (Fig. 1, A). A plasmid is a self-replicating, cir- cular piece of DNA that is used to transfer specific genes into cells. The cDNA plasmid is then digested with restriction enzymes and in- serted into a larger plasmid contain- ing a promoter sequence (among other sequences), thereby creating an expression plasmid (Fig. 1, B). The promoter sequence induces transcription of the cDNA once gene transfer into a cell has oc- curred. Frequently, the promoter is constitutive, meaning it is always “on” and the gene will be tran- scribed continuously until the gene is lost. The cytomegalovirus pro- moter is a commonly used consti- tutive promoter. Investigators are currently developing regulated pro- moters, which can be turned “on” or “off” with an exogenous stimulus (i.e., a drug). Regulated promoters are obviously desirable so that gene expression may occur only at the desired, critical time periods. Once a functional expression plasmid has been created, one must deter- mine the optimal vector with which to transport the expression plasmid safely and efficiently into the de- sired cells. Vectors are vehicles that facili- tate transfer of a particular gene into cells. Vectors are subdivided into viral and nonviral vectors. The most commonly used nonviral vec- tors are liposomes. Liposomes are phospholipid vesicles capable of fusing with a cell membrane, there- by delivering their contents (ex- pression plasmid) into the cell. Liposomes are relatively cheap and nonpathogenic, but their use results in inferior gene-transfer efficiency compared with viral vectors. Other nonviral methods include the gene gun (DNA loaded onto gold beads injected into the cell using a helium “gun”), DNA conjugates (DNA conjugated to certain polycations that improve DNA adherence to cell membranes, thereby improving gene transfer), gene-activated matrices (matrices, or scaffolds, im- pregnated with expression plas- mids), and nonviral-viral hybrids (viral DNA sequences that facilitate longer gene expression or integra- tion into host cell DNA spliced into an expression plasmid). Successful gene transfer using nonviral vectors is termed trans- fection. Alternatively, successful gene transfer using viral vectors is termed transduction. Using current methods, the efficiency of nonviral transfection is generally inferior to that of viral-mediated transduction. Therefore, despite the nonpatho- genic nature and economic advan- tages of nonviral vectors, most cur- rent gene therapy applications use viral vectors. Viruses are highly efficient at infecting cells to deliver genetic material. Various viruses have dif- ferent advantages and disadvan- tages when applied to gene therapy (Table 2). The most commonly used viruses in current gene therapy ap- plications include adenoviruses, retroviruses, herpes simplex viruses (HSVs), and adeno-associated viruses (AAVs). Certain viral DNA se- quences are often removed to ren- der the viral replication defective before gene therapy applications. Adenoviruses have the advan- tage of infecting both dividing and some nondividing cells, thereby achieving a high level of transient gene expression. The adenovirus G E N E restriction sites polyadenylation restriction sites restriction sites cDNA restriction sites A A A G E N E P R O M O T E R A M P N E O EXPRESSION PLASMID Figure 1 A, Complementary DNA (cDNA) consisting of desired gene and restriction enzyme sites in circular form. B, An expression plasmid is created by inserting the desired gene into plasmid containing other sequences. A promoter initiates transcription of the gene, and the polyadenylation sequence helps stabilize the mRNA. Ampicillin (AMP) and neomycin (NEO) resistance genes also may be present to allow in vitro selection of trans- duced or transfected cells. A B Douglas S. Musgrave, MD, et al Vol 10, No 1, January/February 2002 9 genome exists as an episome within the cell’s nucleus, not being inte- grated into the cell’s genome. Low- level production of adenoviral anti- gens often results in an immune response to infected cells, resulting in loss of gene expression after sev- eral weeks. Research is ongoing to develop “gutted” adenoviral vec- tors, whereby the majority of viral protein expression is eliminated to minimize the immune response to adenoviral antigens. Retroviruses are small RNA viruses that, once inside the cell, have their RNA transcribed by the enzyme reverse transcriptase into double-stranded DNA. The double- stranded DNA enters the cell nu- cleus and is integrated, although at a random site, into the cell’s genome. Consequently, the therapeutic gene is expressed for the life of the cell. The disadvantages of retroviral vec- tors are that they infect and trans- duce only actively dividing cells and that their DNA is randomly integrated into the host cell genome. This random integration theoreti- cally can alter oncogene expression and adversely affect cell behavior. HSV is capable of infecting both dividing and nondividing cells, car- rying large amounts of DNA, infect- ing many different cell types, and establishing latency in neuronal cells. First-generation HSV vectors demonstrated toxicity in certain cell types and expressed significant anti- genicity, resulting in only transient gene transfer in certain cell types. Next-generation HSV vectors thus are being engineered to minimize these drawbacks. AAV has the advantages of infecting nondividing cells and of stable integration of its DNA into a specific site on chromosome 19, which appears to be nonpatho- genic. 2 Furthermore, AAV is not known to cause any disease. 3 The main disadvantage of AAV is its capacity to accommodate only small amounts of exogenous DNA, on the order of 5.2 kilobase (kb) pairs. The appropriate viral vector for a given gene therapy application depends on the duration of gene expression desired, the cell type to be transduced, the immune pro- tected or unprotected environment in which the cells reside, and the method chosen to ensure patient safety (i.e., in vitro and/or in vivo safety testing). As mentioned, two fundamental approaches to gene therapy exist: in vivo (direct) and ex vivo (outside the host) (Fig. 2). The in vivo ap- proach consists of directly injecting or implanting the gene-carrying vector into the patient. This ap- proach is attractive for its technical simplicity although it is limited by the inability to perform in vitro safety testing on the transduced cells. The alternative ex vivo approach consists of isolating cells in vitro from a tissue biopsy taken from the patient. The cells are expanded and then transfected or transduced in the laboratory. The genetically altered cells can be tested in vitro for both successful gene transfer and abnormal behavior before they are reimplanted in the patient. After in vitro testing, the cells are introduced into the patient so that they may produce the genetically specified protein. The ability to test for successful gene transfer and abnormal cellular behavior before cell implantation is an advantage of the ex vivo approach. However, the ex vivo method is technically more complex than the in vivo approach. Mesenchymal Stem Cells Growth factors are capable of acting on many different cell types. However, mesenchymal stem cells (MSCs) are the ideal cell type for Table 2 Common Viral Vectors Viral Vector Advantages Disadvantages Adenovirus Infects dividing and nondividing cells Viral antigens can induce immune response Infects many different cell types DNA remains episomal; does not integrate Straightforward vector production Retrovirus DNA integrates into cell’s genome Random DNA integration Accomodates 8 kb Infects only dividing cells Straightforward vector production Herpes simplex virus Infects dividing and nondividing cells Toxicity Accomodates >35 kb Transient expression in certain cell types Adeno-associated virus Infects nondividing cells Accomodates only 5.2 kb Nonpathogenic Difficult vector production Infects many different cell types Gene Therapy and Tissue Engineering Journal of the American Academy of Orthopaedic Surgeons 10 use in tissue engineering and gene therapy approaches. MSCs are rest- ing stem cells capable of differenti- ation into multiple connective tis- sue lineages (such as muscle, bone, ligament, tendon, and cartilage). 4,5 MSCs are not to be confused with hematopoietic stem cells, which are obtained from fetal umbilical cord blood and are capable of differenti- ation into multiple hematopoietic lineages (such as megakaryocytes, erythrocytes, lymphocytes). 6 The existence of MSCs facilitates seemingly endless tissue engineering possibilities. Theoretically, an MSC could be stimulated to undergo dif- ferentiation down a desired lineage, thereby recreating a certain tissue for therapeutic use. Isolation of fully differentiated cells (i.e., chondro- cytes) for a certain tissue engineering application can be hindered by donor site morbidity and limited cell availability. The use of MSCs would obviate the need to obtain fully dif- ferentiated cells for a specific tissue engineering application. MSCs are thought to reside in the bone marrow and thus are obtained by bone marrow biopsy. Resting osteoprogenitor stem cells reside in the bone marrow stroma, and bone marrow–derived MSCs have been used in animal studies to heal carti- lage defects 7 as well as produce cel- lular elements that could modify the clinical course of muscle diseases. 8 Additionally, the feasibility of using MSCs in gene therapy has been de- monstrated in animal models. 9 Furthermore, the effective human clinical use of MSCs to treat neo- nates with osteogenesis imperfecta has been reported. 10,11 Clinical im- provement was demonstrated in neonates with osteogenesis imper- fecta who received allogeneic bone marrow transplantation aimed at replacing their abnormal MSCs. MSCs also can reside outside the bone marrow: evidence supports the existence of resting stem cells in skeletal muscle that are capable of either myogenic or osteogenic dif- ferentiation. 12-14 The availability of skeletal muscle and the relative ease of cell isolation make skeletal mus- cle an attractive source of potential stem cells. The relationship of these muscle-derived stem cells and MSCs still is unclear and remains to be in- vestigated fully. In addition, stem cells possibly may reside in other tissues, such as skin, brain, kidney, or perivascular tissue. Further research must be conducted to elucidate fully the existence and residence of stem cells and to refine efficient isolation tech- niques. Scaffolds A scaffold to support tissue growth is the final component of any tissue engineering approach. This scaffold can take the form of in situ host tissue (e.g., meniscus), transplanted host tissue (e.g., skele- tal muscle flap), naturally derived polymers (such as collagen and hyaluronic acid), synthetic poly- mers (such as poly[L-lactic acid] [PLLA], polyglycolic acid [PGA], and poly[ DL-lactic-co-glycolic acid] [PLGA]), or injectable polymers that cross-link in situ (such as algi- nate and polyethylene oxide [PEO]) (Table 3). Scaffolds influence cell recruitment, cell containment, cell adherence, diffusion of nutrients to the cells, delivery of growth fac- tors, and cell behavior. An ideal scaffold provides for uniform dis- tribution of cells throughout its three-dimensional lattice, facilitates efficient diffusion of biochemical molecules, and undergoes degrada- tion at the same rate that it can be replaced by host tissue. The latter characteristic dictates the species- specific suitability of a scaffold. Certain scaffolds can be impreg- nated with expression vectors, there- by creating gene-activated matrices (GAMs). In addition to fulfilling the previously mentioned scaffold char- acteristics, GAMs can provide for nonviral direct gene transfer, thereby avoiding the immunologic risks of Figure 2 Two basic approaches of gene therapy exist. Ex vivo gene therapy consists of cell harvest and expansion in culture, in vitro transduction or transfection of the isolated cells using the appropriate vectors, and reimplantation of the transduced cells into the patient. In vivo gene therapy consists of direct injection of the vectors into the patient. EX VIVO IN VIVO Cell harvest and culture In vitro transduction Virus Reimplantation of transduced cells Direct injection of vector Douglas S. Musgrave, MD, et al Vol 10, No 1, January/February 2002 11 viral vectors. Host-tissue scaffolds are attractive because they obviate the need for a foreign body, may contain responsive cells, and poten- tially can be vascularized. However, donor-site morbidity and lack of available tissue are prohibitive fac- tors. Naturally derived polymers are attractive because they may be remodeled by host cells to provide space for growing tissue. Collagen menisci constructed from bovine Achilles tendon type I collagen were implanted into eight patients, with encouraging results at second-look arthroscopy and 2-year clinical follow-up. 15 The drawbacks of natu- rally derived polymers are ensuring pathogen removal and potential lim- ited supply. Synthetic polymers, in contrast, can be mass-produced and de- signed specifically for a particular application. Their molecular com- position can be modified to affect scaffold-cell interactions and scaf- fold degradation. The presence of a foreign body, however, may result in an inflammatory reaction, to the detriment of tissue growth, and increase the risk of infection. Syn- thetic polymers have been used to create composite phalanges by seeding bovine chondrocytes and tenocytes onto sheets of PGA and suturing these structures to bovine periosteum wrapped around cova- lently linked PLLA and PGA. 16 The composite structures were subcuta- neously implanted into athymic mice to facilitate growth. No gene transfer was used. After 20 weeks, the composite tissue resembled the gross and histologic appearance of human phalanges. The science of polymer chemistry governing the porosity, cell- and growth factor–binding characteris- tics, and degradation kinematics of synthetic polymers is beyond the scope of this article. It must be noted, however, that, given each scaffold’s inherent advantages and disadvan- tages, the scaffold for a given tissue engineering application must be cho- sen carefully. Specific Tissues Bone The existence of bone-inducing growth factors has long been rec- ognized. 17 These bone-inducing growth factors, termed bone mor- phogenetic proteins (BMPs), are members of the transforming growth factor-β (TGF-β) superfamily. Recom- binant human BMP-2 (rhBMP-2) 18 has been implanted directly on var- ious carriers and can heal critically sized bone defects in animal mod- els. 19 Likewise, other BMPs, such as BMP-3 20 and BMP-7 (osteogenic protein-1), 21 have proved to be effica- cious in animal models. To achieve sustained BMP delivery, investiga- tors have developed both in vivo and ex vivo gene therapy approaches using BMPs. Direct adenovirus- mediated approaches delivering BMP-2 22,23 and BMP-9, 24 as well as a direct GAM-mediated approach delivering BMP-4, 25 have been reported. Ex vivo approaches to deliver BMP-2 have used rodent bone marrow stromal cell lines, 26,27 primary rodent bone marrow stro- mal cells, 28,29 primary rodent skele- tal muscle–derived cells, 12,29 primary human skeletal muscle–derived cells, 30 primary rabbit articular chon- drocytes, 29 primary rabbit perios- teal cells (BMP-7), 31 and primary rabbit skin fibroblasts. 29 For clin- ical applications, primary autolo- gous cells (cells isolated from the host) are preferable to cell culture lines, which are allogeneic or xeno- geneic and may have tumorigenic and immunogenic potential. The direct, in vivo gene therapy approach using BMPs has been ap- plied in rodent spine fusion mod- els, 22,24 whereas the ex vivo ap- proach has been applied to healing critically sized rodent bone de- fects. 13,26,28 In the spine fusion Table 3 Categories of Scaffolds Type of Scaffold Advantages Disadvantages Host tissue Responsive cells Donor site morbidity (e.g., meniscus, skeletal muscle flap) Viability and potential vascularity Limitation on amount of tissue No foreign body Naturally derived polymers Remodeling potential Pathogen removal must be assured (e.g., collagen, hyaluronic acid) No donor site morbidity Potential limited supply Synthetic polymers Mass production Foreign body (infection risk) (e.g., PLLA, PGA, PLGA) Custom designed Possible inflammatory reaction Injectable synthetic polymers Mass production Foreign body (infection risk) (e.g., alginate, PEO) Can fill complex spaces In vivo chemical reaction Gene Therapy and Tissue Engineering Journal of the American Academy of Orthopaedic Surgeons 12 models, the adenoviral vector was directly injected into the paraspinal musculature, 22,24 resulting in para- spinal bone formation present at 3 weeks after injection. In the ex vivo approaches, the transduced cells were seeded onto collagen sponges 13 or demineralized bone matrix 26,28 as scaffolds, resulting in improved radiographic healing of both calvar- ial 13 and long bone defects. 26,28 A novel osteoinductive protein termed LIM mineralization protein- 1 (LMP-1) has been used in an ex vivo gene therapy model for spine fusions. 32 Because LMP-1 is an intracellular signaling molecule (regulated by BMP-6), the use of LMP-1 requires gene transfer. Bone marrow stromal cells transfected with LMP-1, seeded onto a scaffold of nonosteogenic devitalized bone matrix, and implanted into the paraspinal area of rats resulted in a 100% rate of spine fusion compared with no fusion in the controls. 32 Which cell type to use in ex vivo gene transfer of osteogenic proteins remains unresolved. The ideal cell type is expendable, easily isolated from the patient, and amenable to efficient transduction and protein secretion; possesses osteocompe- tence 17 ; and has favorable survival characteristics when reimplanted into the patient. Bone marrow stro- mal cells 26,28,29 and skeletal mus- cle–derived cells 12-14,29 both possess osteocompetence and have been used successfully in ex vivo osteo- genic protein gene transfer. Fi- nally, as mentioned previously, the development of composite pha- langes may be possible without gene therapy using a combination of cell transplantation and polymer scaffolds. 16 Growth plate disorders are an- other possible skeletal gene therapy application. In a rabbit physeal injury model, direct adenovirus- mediated gene transfer of insulin- like growth factor-1 (IGF-1) was shown to inhibit subsequent phy- seal growth disturbance, whereas direct BMP-2 gene transfer promoted premature physeal closure and sub- sequent growth disturbance. 33 These results warrant further investigation into the use of IGF-1 gene transfer to inhibit physeal closure after physeal fractures and into the development of physiodesis using BMP-2 gene transfer. Articular Structures Patients with a variety of articu- lar disorders are potential candi- dates for gene therapy and tissue engineering applications. Innova- tive approaches for treating arthri- tis, chondral and osteochondral de- fects, meniscal tears, and ligament injuries currently are being investi- gated. Arthritis Arthritis was the first nonlethal disease for which a human gene therapy trial was approved and undertaken. 34 This trial consisted of the ex vivo transfer of the inter- leukin-1 (IL-1) receptor antagonist protein (IRAP or IL-1RA) to syno- viocytes obtained from six post- menopausal women with severe rheumatoid arthritis. A retroviral vector was used to transduce the synoviocytes with IRAP, a protein that inhibits the arthritogenic cy- tokine IL-1. The transduced syn- oviocytes were injected into the patients’ metacarpophalangeal (MCP) joints 1 week before sched- uled MCP joint arthroplasty. At the time of MCP joint arthroplasty, the joints, synovium, and synovial fluid were harvested for analysis. The trial was designed to establish feasi- bility and safety but not necessarily efficacy. No adverse reactions in the patients have been reported to date, and lifelong clinical follow-up continues. Animal studies have established that direct adenoviral-mediated gene transfer of a soluble IL-1 recep- tor and a soluble tumor necrosis factor-α receptor reduce cartilage matrix degradation in rabbit knees with antigen-induced arthritis. 35 Combining multiple antiarthritic proteins in gene therapy applica- tions may lead to additive or syner- gistic effects. Cartilage Different approaches have been applied to chondral and osteochon- dral defects. RhBMP-2 regulates the behavior of costochondral growth plate chondrocytes 36 and maintains the phenotype of articular chon- drocytes in cell culture. 37 Collagen sponges impregnated with rhBMP-2 improve the healing of rabbit full- thickness cartilage defects. 38 The feasibility of using articular chon- drocytes in ex vivo BMP-2 gene transfer has been established. 16 The effects of in vitro gene transfer of IGF-1, BMP-2, and TGF-β to rabbit articular chondrocytes have recently been investigated. 39 Gene transfer of BMP-2 was a potent stimulus for proteoglycan synthesis in the pres- ence of IL-1. Gene transfer of IGF-1 was a strong stimulus for collagen and noncollagenous protein synthe- sis. Other factors, such as BMP-7, cartilage growth and differentiation factors, and various transcription factors, also may play a role carti- lage healing. 40 Cell transplantation of autolo- gous chondrocytes injected into chondral defects and covered with a periosteal flap has shown some promise clinically, especially for femoral condyle lesions. 41 Cell transplantation using various natu- rally derived scaffolds, synthetic scaffolds, injectable scaffolds, and autologous scaffolds 42 is being in- vestigated in animal models. 43 In addition to other tissues, bio- reactor vessels may hold promise for tissue engineered cartilage. Bioreactor vessels are cell culture containers specially designed to facilitate manipulation of the cells’ environment. Environmental fac- Douglas S. Musgrave, MD, et al Vol 10, No 1, January/February 2002 13 tors, such as culture medium flow and mechanical stress, can be ma- nipulated to influence cell behavior and tissue growth. Bioreactor ves- sels can be used both to engineer cartilage for possible implantation and provide a controlled in vitro environment for the study of chon- drogenesis. 44 Size limitations of bioreactor vessels are the current restraints to clinical application. The optimal combination of growth factor, delivery method, cells, and scaffold to heal cartilage injuries is the subject of ongoing investigation. Meniscus The possibility of creating in vitro a custom replacement menis- cus using scaffolds, 15 cells, and/or gene therapy for subsequent in vivo implantation is intriguing. Alterna- tively, meniscal cells might be mod- ulated in vivo using gene therapy to promote healing of certain injuries. Meniscal cells are amenable to gene transfer of both marker genes and various growth factor genes using either in vivo or ex vivo gene ther- apy, with gene expression persisting for up to 6 weeks. 45-47 Successful ex vivo gene transfer has been accom- plished using either myoblasts 46,47 or meniscal cells. 45 Implanted me- niscal scaffolds constructed of bo- vine collagen appear to be replaced by host tissue mimicking a human meniscus. 15 Research is ongoing into the preferred growth factors to promote meniscal healing, tech- niques to improve long-term gene expression, and the optimal scaffold needed to create new menisci. Ligaments Gene therapy techniques also are being applied to ligaments. β- Galactosidase (lacZ) gene transfer to ligament has proved to be feasible in animal models by either the in vivo or ex vivo approach, using both ade- novirus as well as retrovirus. Ex vivo gene transfer to ligaments has been successfully achieved using either ligament fibroblasts 48,49 or skeletal muscle myoblasts. 49 Many growth factors, such as basic fibro- blast growth factor, platelet-derived growth factor (PDGF), vascular endothelial growth factor, IGF-1 and -2, TGF-β, and BMP-12, may play roles in ligament healing. Data sug- gest that PDGF stimulates cell divi- sion and migration, whereas TGF-β and the IGFs promote extracellular matrix synthesis. 50 Direct gene transfer of PDGF-β using a viral- liposome conjugate vector into rat patellar ligament resulted in initial improved angiogenesis and subse- quent enhanced extracellular matrix synthesis. 51 Studies to improve liga- ment-bone healing using BMP-12 and to engineer ligament grafts in vitro are ongoing. Intervertebral Disk Intervertebral disk nucleus pul- posus cells reside in an immuno- privileged site, which makes them potentially attractive targets for gene therapy approaches. Direct adenovirus-mediated gene transfer of lacZ into rabbit nucleus pulposus cells in an in vivo model resulted in persistent gene expression for at least 12 weeks. 52 A similar approach has been used to transfer the human TGF-β1 gene to rabbit nucleus pul- posus cells in vivo. 53 The cells were harvested 1 week later and in vitro assays were performed. TGF-β1 gene transfer resulted in a 30-fold increase in active TGF-β1, a fivefold increase in total TGF-β1, and a 100% increase in proteoglycan synthesis. The results suggest that gene ther- apy to treat degenerative disk dis- ease warrants further investigation. Skeletal Muscle Both in vivo and ex vivo forms of gene therapy to skeletal muscle for various types of inherited diseases, such as Duchenne muscular dystro- phy, have been investigated for many years. 54 In clinical trials, autol- ogous myoblast transplantation for Duchenne muscular dystrophy has proved to be safe, 55 although myo- blast survival is often suboptimal for inherited muscle diseases. 54 Additionally, ongoing research into novel growth factors that might facilitate gene therapy approaches to expedite the healing of acquired muscle injury has many potentially far-reaching applications. IGF-1, basic fibroblast growth factor, and nerve growth factor have been shown in rodent models to improve muscle healing and strength (fast- twitch or tetanic strength) after con- tusion, laceration, and strain. 56-58 However, myoblast transplantation combined with ex vivo gene therapy is an even more attractive approach that can be used to deliver appropri- ate growth factors and responsive cells for acquired, traumatic muscle injuries. Summary The biologic era of orthopaedic surgery promises to change the care of musculoskeletal disorders in this century. Extensive laboratory in- vestigations and preliminary clinical investigations have established the feasibility and promise of gene ther- apy and tissue engineering. Re- search must continue to further un- derstanding of cytokines, gene delivery vectors, MSCs, and scaf- folds. Pertinent issues, such as the optimal growth factors or genes, the timing and control of growth factor delivery, the optimal target cell, and the effectiveness of various scaf- folds, must be addressed before widespread clinical use can occur. Most important, early clinical trials must establish patient safety before clinical efficacy is sought. Respon- sible and innovative investigation will lead orthopaedic surgery into this new biologic era. Gene Therapy and Tissue Engineering Journal of the American Academy of Orthopaedic Surgeons 14 References 1. Huard J, Fu FH (eds): Gene Therapy and Tissue Engineering in Orthopaedic and Sports Medicine. Boston, MA: Birkhäuser, 2000. 2. Xiao X, Pruchnic R, Huard J: Adeno- associated virus (AAV) vectors for musculoskeletal gene transfer, in Huard J, Fu FH (eds): Gene Therapy and Tissue Engineering in Orthopaedic and Sports Medicine. Boston, MA: Birkhäuser, 2000, pp 260-273. 3. Berns KI, Bohenzky RA: Adeno-asso- ciated viruses: An update. Adv Virus Res 1987;32:243-306. 4. Caplan AI: Mesenchymal stem cells and gene therapy. Clin Orthop 2000; 379(suppl):S67-S70. 5. Pittenger MF, Mackay AM, Beck SC, et al: Multilineage potential of adult human mesenchymal stem cells. Science 1999;284:143-147. 6. Smith C, Storms B: Hematopoietic stem cells. Clin Orthop 2000;379(suppl): S91-S97. 7. Grande DA, Southerland SS, Manji R, Pate DW, Shwartz RE, Lucas PA: Repair of articular cartilage defects using mesenchymal stem cells. Tissue Eng 1995;1:345-353. 8. Saito T, Dennis LE, Lennon DP, Young RG, Caplan AI: Myogenic expression of mesenchymal stem cells within myotubes of mdx mice in vitro and in vivo. Tissue Eng 1995;1:327-344. 9. Allay JA, Dennis JE, Haynesworth SE, et al: LacZ and IL-3 expression in vivo after retroviral transduction of mar- row-derived human osteogenic mes- enchymal progenitors. Hum Gene Ther 1997;8:1417-1427. 10. Horwitz EM, Prockop DJ, Fitzpatrick LA, et al: Transplantability and thera- peutic effects of bone marrow-derived mesenchymal cells in children with osteogenesis imperfecta. Nat Med 1999;5:309-313. 11. Horwitz EM, Prockop DJ, Gordon PL, et al: Clinical responses to bone mar- row transplantation in children with severe osteogenesis imperfecta. Blood 2001;97:1227-1231. 12. Bosch P, Musgrave DS, Lee JY, et al: Osteoprogenitor cells within skeletal muscle. J Orthop Res 2000;18:933-944. 13. Lee JY, Musgrave D, Pelinkovic D, et al: Effect of bone morphogenetic pro- tein-2-expressing muscle-derived cells on healing of critical-sized bone defects in mice. J Bone Joint Surg Am 2001;83:1032-1039. 14. Lee JY, Qu-Petersen Z, Cao B, et al: Clonal isolation of muscle-derived cells capable of enhancing muscle regeneration and bone healing. J Cell Biol 2000;150:1085-1100. 15. Rodkey WG, Steadman JR, Li S-T: A clinical study of collagen meniscus im- plants to restore the injured meniscus. Clin Orthop 1999;367(suppl):S281-S292. 16. Isogai N, Landis W, Kim TH, Gersten- feld LC, Upton J, Vacanti JP: Forma- tion of phalanges and small joints by tissue-engineering. J Bone Joint Surg Am 1999;81:306-316. 17. Urist MR: Bone: Formation by autoin- duction. Science 1965;150:893-899. 18. Wang EA, Rosen V, D’Alessandro JS, et al: Recombinant human bone mor- phogenetic protein induces bone for- mation. Proc Natl Acad Sci USA 1990; 87:2220-2224. 19. Zegzula HD, Buck DC, Brekke J, Wozney JM, Hollinger JO: Bone for- mation with the use of rhBMP-2 (recombinant human bone morpho- genetic protein-2). J Bone Joint Surg Am 1997;79:1778-1790. 20. Khouri RK, Brown DM, Koudsi B, et al: Repair of calvarial defects with flap tissue: Role of bone morphogenetic proteins and competent responding tissues. Plast Reconstr Surg 1996;98: 103-109. 21. Cook SD, Wolfe MW, Salkeld SL, Rueger DC: Effect of recombinant human osteogenic protein-1 on heal- ing of segmental defects in non-human primates. J Bone Joint Surg Am 1995; 77:734-750. 22. Alden TD, Pittman DD, Beres EJ, et al: Percutaneous spinal fusion using bone morphogenetic protein-2 gene therapy. J Neurosurg 1999;90(1 suppl):109-114. 23. Musgrave DS, Bosch P, Ghivizzani S, Robbins PD, Evans CH, Huard J: Adenovirus-mediated direct gene therapy with bone morphogenetic pro- tein-2 produces bone. Bone 1999;24: 541-547. 24. Helm GA, Alden TD, Beres EJ, et al: Use of bone morphogenetic protein-9 gene therapy to induce spinal arthro- desis in the rodent. J Neurosurg 2000; 92(2 suppl):191-196. 25. Fang J, Zhu Y-Y, Smiley E, et al: Stim- ulation of new bone formation by direct transfer of osteogenic plasmid genes. Proc Natl Acad Sci USA 1996;93: 5753-5758. 26. Lieberman JR, Le LQ, Finerman GA, Berk A, Witte ON, Stevenson S: Re- gional gene therapy with a BMP-2- producing murine stromal cell line induces heterotopic and orthotopic bone formation in rodents. J Orthop Res 1998;16:330-339. 27. Lou J, Xu F, Merkel K, Manske P: Gene therapy: Adenovirus-mediated human bone morphogenetic protein-2 gene transfer induces mesenchymal progenitor cell proliferation and dif- ferentiation in vitro and bone forma- tion in vivo. J Orthop Res 1999;17:43-50. 28. Lieberman JR, Daluiski A, Stevenson S, et al: The effect of regional gene therapy with bone morphogenetic pro- tein-2-producing bone-marrow cells on the repair of segmental femoral defects in rats. J Bone Joint Surg Am 1999;81:905-917. 29. Musgrave DS, Bosch P, Lee JY, et al: Ex vivo gene therapy to produce bone using different cell types. Clin Orthop 2000;378:290-305. 30. Musgrave DS, Pruchnic RJ, Ziran BH, et al: Abstract: Human muscle-derived cells in ex vivo BMP-2 gene therapy to produce bone. Rosemont, IL: Ameri- can Academy of Orthopaedic Sur- geons, Proceedings of the 67th Annual Meeting, Orlando, Florida, 2000, p 351. 31. Mason JM, Grande DA, Barcia M, Grant R, Pergolizzi RG, Breitbart AS: Expression of human bone morpho- genetic protein 7 in primary rabbit periosteal cells: Potential utility in gene therapy for osteochondral repair. Gene Ther 1998;5:1098-1104. 32. Boden SD, Titus L, Hair G, et al: Lumbar spine fusion by local gene therapy with a cDNA encoding a novel osteoinductive protein (LMP-1). Spine 1998;23:2486-2492. 33. Lee C, Martinek V, Musgrave DS, et al: Abstract: Muscle-based gene therapy and tissue engineering for tibial phy- seal defects. Trans Orthop Res Soc 2000;25:1072. 34. Evans CH, Robbins PD, Ghivizzani SC, et al: Clinical trial to assess the safety, feasibility, and efficacy of trans- ferring a potentially anti-arthritic cytokine gene to human joints with rheumatoid arthritis. Hum Gene Ther 1996;7:1261-1280. 35. Ghivizzani SC, Lechman ER, Kang R, et al: Direct adenovirus-mediated gene transfer of interleukin 1 and tumor necrosis factor alpha soluble receptors to rabbit knees with experi- mental arthritis has local and distal anti-arthritic effects. Proc Natl Acad Sci USA 1998;95:4613-4618. Douglas S. Musgrave, MD, et al Vol 10, No 1, January/February 2002 15 36. Erickson DM, Harris SE, Dean DD, et al: Recombinant bone morphogenetic protein (BMP)-2 regulates costochon- dral growth plate chondrocytes and induces expression of BMP-2 and BMP-4 in a cell maturation-dependent manner. J Orthop Res 1997;15:371-380. 37. Sailor LZ, Hewick RM, Morris EA: Recombinant human bone morpho- genetic protein-2 maintains the artic- ular chondrocyte phenotype in long- term culture. J Orthop Res 1996;14: 937-945. 38. Sellers RS, Peluso D, Morris EA: The effect of recombinant human bone morphogenetic protein-2 (rhBMP-2) on the healing of full-thickness defects of articular cartilage. J Bone Joint Surg Am 1997;79:1452-1463. 39. Smith P, Shuler FD, Georgescu HI, et al: Genetic enhancement of matrix synthesis by articular chondrocytes: Comparison of different growth factor genes in the presence and absence of interleukin-1. Arthritis Rheum 2000;43: 1156-1164. 40. Evans CH, Ghivizzani SC, Smith P, Shuler FD, Mi Z, Robbins PD: Using gene therapy to protect and restore cartilage. Clin Orthop 2000;379(suppl): S214-S219. 41. Brittberg M, Lindahl A, Nilsson A, Ohlsson C, Isaksson O, Peterson L: Treatment of deep cartilage defects in the knee with autologous chondrocyte transplantation. N Engl J Med 1994; 331:889-895. 42. Lee CW, Fukushima K, Usas A, et al: Abstract: Myoblast mediated gene therapy with muscle as a biological scaffold for the repair of full-thickness defects of articular cartilage. Trans Orthop Res Soc 2000;25:1068. 43. Temenoff JS, Mikos AG: Review: Tissue engineering for regeneration of articular cartilage. Biomaterials 2000; 21:431-440. 44. Freed LE, Martin I, Vunjak-Novakovic G: Frontiers in tissue engineering: In vitro modulation of chondrogenesis. Clin Orthop 1999;367(suppl):S46-S58. 45. Goto H, Shuler FD, Lamsam C, et al: Transfer of lacZ marker gene to the meniscus. J Bone Joint Surg Am 1999; 81:918-925. 46. Day CS, Kasemkijwattana C, Menetrey J, et al: Myoblast-mediated gene trans- fer to the joint. J Orthop Res 1997;15: 894-903. 47. Kasemkijwattana C, Menetrey J, Goto H, Niyibizi C, Fu FH, Huard J: The use of growth factors, gene therapy and tissue engineering to improve meniscal healing. Materials Science and Engineering C 2000;13:19-28. 48. Hildebrand KA, Deie M, Allen CR, et al: Early expression of marker genes in the rabbit medial collateral and anterior cruciate ligaments: The use of different viral vectors and the effects of injury. J Orthop Res 1999;17:37-42. 49. Menetrey J, Kasemkijwattana C, Day CS, et al: Direct-, fibroblast- and myoblast-mediated gene transfer to the anterior cruciate ligament. Tissue Eng 1999;5:435-442. 50. Evans CH: Cytokines and the role they play in the healing of ligaments and tendons. Sports Med 1999;28:71-76. 51. Nakamura N, Shino K, Natsuume T, et al: Early biological effect of in vivo gene transfer of platelet-derived growth factor (PDGF)-B into healing patellar ligament. Gene Ther 1998;5: 1165-1170. 52. Nishida K, Kang JD, Suh J-K, Robbins PD, Evans CH, Gilbertson LG: Adenovirus-mediated gene transfer to nucleus pulposus cells: Implications for the treatment of intervertebral disc degeneration. Spine 1998;23:2437-2443. 53. Nishida K, Kang JD, Gilbertson LG, et al: Modulation of the biologic activity of the rabbit intervertebral disc by gene therapy: An in vivo study of adeno- virus-mediated transfer of the human transforming growth factor β1 encod- ing gene. Spine 1999;24:2419-2425. 54. Huard J, Acsadi G, Jani A, Massie B, Karpati G: Gene transfer into skeletal muscles by isogenic myoblasts. Hum Gene Ther 1994;5:949-958. 55. Huard J, Roy R, Bouchard JP, Malouin F, Richards CL, Tremblay JP: Human myoblast transplantation between immunohistocompatible donors and recipients produces immune reactions. Transplant Proc 1992;24:3049-3051. 56. Menetrey J, Kasemkijwattana C, Fu FH, Moreland MS, Huard J: Suturing versus immobilization of a muscle lac- eration: A morphological and func- tional study in a mouse model. Am J Sports Med 1999;27:222-229. 57. Kasemkijwattana C, Menetrey J, Bosch P, et al: Use of growth factors to im- prove muscle healing after strain injury. Clin Orthop 2000;370;272-285. 58. Kasemkijwattana C, Menetrey J, Somogyl G, et al: Development of ap- proaches to improve the healing follow- ing muscle contusion. Cell Transplant 1998;7:585-598. . potent stimulus for proteoglycan synthesis in the pres- ence of IL-1. Gene transfer of IGF-1 was a strong stimulus for collagen and noncollagenous protein synthe- sis. Other factors, such as BMP-7, cartilage