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© 1999 The Journal of Bone and Joint Surgery, Inc. Transfer of LacZ Marker Gene to the Meniscus*  , PITTSBURGH, PENNSYLVANIAInvestigation performed at Ferguson Laboratory, University of Pittsburgh School of Medicine, Pittsburgh
Background: Lesions in the avascular two-thirds of the meniscus do not heal well and are of concern clinically. Various growth factors promote the synthesis of matrix by meniscal cells and thus have the potential to augment healing. However, their clinical application is severely hindered by problems with delivery. An attractive approach to overcoming such problems is to transfer genes that encode the growth factors in question to the site of the injury. As a prelude to this, we evaluated methods for delivering genes to the meniscus. Methods: Gene transfer was evaluated in vitro and in vivo with a lacZ marker gene, which expresses the enzyme ß-galactosidase. Two types of vectors were tested: an adenovirus and a retrovirus. Monolayers of lapine, canine, and human meniscal cells, as well as intact lapine and human menisci, were used for the in vitro studies. Lesions were created in the menisci of rabbits and dogs for the in vivo studies. Gene transfer to the sites of the experimental meniscal lesions in vivo was accomplished in two ways. In the lapine model, a suspension of adenovirus carrying the lacZ marker gene was mixed with whole blood and the clot was inserted into the lesion. In the canine model, retrovirally transduced allogenic meniscal cells carrying the lacZ marker gene were embedded in collagen gels and transferred to the defects. The animals were killed at various time-points, and gene expression was evaluated by histological examination of sections stained with 5-bromo-4-chloro-indolyl-ß-D-galactose (X-gal), from which a blue chromagen is released in the presence of ß-galactosidase. Results: Monolayer cultures of lapine, canine, and human meniscal cells were susceptible to genetic transduction by both adenoviral and retroviral vectors. In vitro gene transfer to intact human and lapine menisci proved possible both by direct, adenoviral, delivery and indirect, retroviral, delivery. Gene expression persisted for at least twenty weeks under in vitro conditions. With regard to the in vivo studies, gene expression persisted within the clot and in some of the adjacent meniscal cells for at least three weeks in the lapine defect model. In the canine defect model, gene expression persisted within the transplanted, transduced meniscal cells for at least six weeks. Conclusions: It is possible to transfer genes to sites of meniscal damage and to express them locally within the lesion for several weeks. Clinical Relevance: Healing of the avascular portion of the meniscus may be improved by the transfer of genes encoding the appropriate growth factors. To our knowledge, the present report is the first to describe methods for transferring genes to the meniscus. When used in conjunction with the appropriate growth-factor genes, these techniques should help to provide the basis for potential alternative treatment options for meniscal lesions. Additional studies are needed to determine whether these techniques will lead to improved healing of meniscal defects in vivo.
The menisci serve many important biomechanical functions, including load-bearing, shock absorption, and load transmission. They also contribute to the stability of the knee14. Because of a greater understanding of the importance of the menisci and many reports demonstrating an early onset of degenerative changes in the knee joint following meniscectomy4,6,20, orthopaedic surgeons now aim to preserve as much functional meniscal tissue as possible when treating meniscal injuries. Because the periphery of the meniscus receives a rich blood supply, tears in this location are reparable. In contrast, the inner two-thirds of the meniscus is relatively avascular. The results of meniscal repair in this zone are variable, although many techniques have been developed in attempts to promote healing in this region2,7,15,24. One approach is to use growth factors, which accelerate the healing process12. It has been demonstrated that meniscal fibrochondrocytes are capable of enhanced proliferation and matrix synthesis when exposed to growth factors that are normally present in wound hematomas3,13,19,21,22. Moreover, animal studies have shown that the addition of growth factors during conventional methods of meniscal repair improves healing9. For this approach to be successful, a method is needed with which to deliver the growth factors locally to the lesion and to maintain the sustained release of the growth factors over time. Direct application of growth factors by themselves is not likely to be successful because of their short biological half-lives. The implantation of bioresorbable slow-release devices, while possible, has yet to be realized in a clinically useful manner. Gene transfer provides a promising alternative strategy5. Delivery to the meniscus of a gene encoding the growth factor of interest may make it possible to achieve sustained local endogenous synthesis of the growth factor at the site of healing. There are many advantages to this approach, including the potential for regulating both the level and the duration of growth-factor production5. Endogenous synthesis also ensures that, unlike recombinant growth factors produced by prokaryotic cells, the growth factor has undergone appropriate posttranslation modification. The present study was designed to evaluate the feasibility of gene transfer to the meniscus. There are two main strategies for transferring genes to sites in the body5. With in vivo, or direct, gene delivery, the new genetic material is introduced directly into the patient. With ex vivo, or indirect, gene delivery, cells are removed, genetically manipulated outside the body, and then returned to the body. The cells used in ex vivo gene delivery do not necessarily have to be derived from the target organ. Gene delivery is facilitated by the use of vectors, which enable the cellular uptake and expression of the transferred genes. A variety of viral and nonviral vectors are available for this purpose17. In the present study, we elected to evaluate vectors developed from an adenovirus and a retrovirus. Adenoviruses infect both dividing and nondividing cells very efficiently and may be used for both in vivo and ex vivo gene delivery. In contrast, retroviruses only transduce dividing cells and are predominantly used in ex vivo protocols. In both cases, the viruses in the present study were used to transfer a bacterial marker gene, lacZ, which expresses the enzyme ß-galactosidase. Cells expressing this enzyme, which is not normally found in mammalian cells, can be stained blue by a simple histochemical procedure.
Viral Vectors The retroviral vector was based on the Moloney murine leukemia virus. It contained the lacZ gene under the control of the retroviral 5' long terminal repeat and the neomycin phosphotransferase gene under the control of the simian virus 40 early promoter16. The latter gene renders cells resistant to the neomycin analog G418. This virus was generated at titers of approximately 105 infectious viral particles per milliliter from a producer line known as CRIP.
Meniscal Cell Culture
Cell Transduction in Vitro Retroviral transduction: Confluent cultures were trypsinized and subcultured at a 1:3 split ratio. When the first-passage cultures were 60 percent confluent, the medium was removed, the cells were washed with Gey's balanced salt solution as described, and one milliliter of retroviral vector suspension was added in the presence of eight micrograms of polybrene (Sigma Chemical, St. Louis, Missouri) per milliliter. This provided a ratio of 5 x 106 viral particles per million cells. The cells were incubated for three hours, with gentle shaking every thirty minutes. After the three hours of incubation, three milliliters of fresh medium was added and the cells were returned to the incubator. When the cells were confluent, they were trypsinized and replated and 0.25 milligram of G418 (Sigma) per milliliter was added into the culture medium to select for transduced cells11. Cells that expressed the neomycin phosphotransferase gene contained in the retrovirus were resistant to the cytotoxic actions of G148. To detect lacZ gene expression by the transduced cells, the cultures were histochemically stained blue with the chromogenic substrate 5-bromo-4-chloro-indolyl-ß-D-galactose (X-gal; Sigma), as described previously16, at every passage. The percentage of lacZ+ cells was determined by scoring cells as positive (blue) or negative (colorless) histologically. At least 100 cells were scored in each case.
In Vitro Gene Transfer to Menisci For direct gene delivery, 0.1 milliliter of the adenoviral vector suspension was injected into the menisci or meniscal fragments at four locations with use of a one-milliliter syringe and a 25-gauge needle. Four menisci or meniscal fragments were injected at each time-point (at one, two, eight, twelve, sixteen, and twenty weeks after the gene transfer). For indirect gene delivery to the menisci or meniscal fragments, cultured meniscal cells were transduced with the retrovirus containing the lacZ and neomycin phosphotransferase genes and were selected in G418 as described earlier. Monolayer cultures of cells were then trypsinized and centrifuged at 2000 revolutions per minute for ten minutes. The cells were collected and were suspended in 0.5 milliliter of Ham's F-12 medium with 10 percent fetal bovine serum and 1 percent penicillin and streptomycin to a final concentration of 106 cells per milliliter. One hundred microliters (105 cells) of the cell suspension was injected at multiple locations into the procured meniscal tissue with a one-milliliter syringe and a 25-gauge needle. After the injections, the intact lapine and fragmented human menisci were placed in Petri dishes that contained the culture medium that lacked G418, and they were maintained in the incubator. The meniscal tissues were fixed sequentially, at one, two, four, eight, twelve, sixteen, and twenty weeks after the gene transfer, with 0.01-molar phosphate-buffered saline solution (Sigma) containing 4 percent paraformaldehyde (Fisher Scientific, Fair Lawn, New Jersey). The meniscal tissue was stained with X-gal to detect gene expression by the transduced cells. The stained menisci were then cut along the transverse plane through the site of the injection. Tissue specimens were dehydrated and embedded in paraffin wax. Paraffin sections were cut at a thickness of five micrometers and were affixed to glass slides. Deparaffinized and hydrated sections were stained with eosin as a counterstain for X-gal.
In Vivo Gene Transfer to Operatively Created Meniscal Lesions
Adenovirally Mediated ex Vivo Gene Transfer to Lapine Meniscal Lesions
Retrovirally Mediated ex Vivo Gene Transfer to Canine Meniscal Lesions Twelve adult mongrel dogs that weighed twenty-five to thirty kilograms were used. All of the dogs had a normal gait and a full range of motion of the limbs. General anesthesia was given and endotracheal intubation was used. Inhalation anesthesia was maintained with a mixture of isoflurane, nitrous oxide, and oxygen. The hindlimbs were shaved, prepared with iodine, and draped in a sterile fashion. A medial parapatellar skin incision was made through the subcutaneous tissue down to the joint capsule. The capsule was dissected to expose the medial collateral ligament. This ligament was detached at the femoral insertion and was reflected distally to expose the medial meniscus. A partial-thickness circular defect, two millimeters in diameter and three millimeters deep, was made three millimeters from the meniscosynovial junction in the middle third of the meniscus. The collagen gel with the transduced canine cells was implanted into the defect. A collagen gel without cells was implanted in the contralateral meniscus to serve as a control. After transplantation, the medial collateral ligament was reattached at the site of its original insertion with interosseous sutures. The capsule was closed carefully, and the skin was closed with a subcuticular stitch. The animals were mobilized postoperatively, given a standard diet, and allowed to walk outside the cages at one week after the operation. LacZ gene expression in the menisci was evaluated at one, two, four, and six weeks postoperatively. Three animals were killed at each time-period. The menisci were obtained, fixed with 0.01-molar phosphate-buffered saline solution that contained 4 percent paraformaldehyde, and stained with X-gal. The stained menisci were cut along the radial plane through the defect and were embedded, sectioned, and stained as described earlier.
Our studies of transfer of the lacZ marker gene to the meniscus comprised three parts: in vitro transduction of cultured cells with adenoviral and retroviral vectors, direct and indirect in vitro delivery of the lacZ marker gene with adenovirus and retrovirus to procured menisci, and ex vivo delivery of the lacZ marker gene with adenovirus and retrovirus to operatively created meniscal lesions in the knee joints of rabbits and dogs.
In Vitro Gene Transfer to Monolayer Cultures After the lapine, canine, and human meniscal cells were transduced with this retrovirus, expression of the lacZ gene was seen initially in 20, 18, and 10 percent of cells, respectively (Table I). Selection of transduced cells in G418 increased the percentage of transduced cells to nearly 100 (Table I). Transgene expression after transduction with the retrovirus and selection in G418 was stable and lasted twenty-eight weeks in vitro, at which time the experiment was terminated. During this twenty-eight-week period, the cells were taken through approximately fifteen passages, without reduction in the percentage of cells expressing lacZ. These experiments were repeated five times with the same results.
Lapine, canine, and human meniscal cells were also transduced with an adenovirus carrying the lacZ gene. At a multiplicity of infection of fifty, nearly 100 percent of the cells of each species expressed the lacZ gene after transduction with this virus (Table II). Gene expression after adenoviral transduction was stable for all species for twenty weeks in confluent, nondividing cultures, but, unlike expression after retroviral transduction, it was not stable on passage (Table II).
In Vitro Gene Transfer to Menisci
Direct Gene Transfer Whole lapine menisci and fragments of human menisci were used to assess the feasibility of direct viral gene transfer with the adenoviral vector carrying the lacZ gene. In vitro injection of this vector directly into lapine menisci led to the transduction of cells adjacent to the track of the needle but not elsewhere, suggesting that the dense, meniscal matrix impeded diffusion of the virus. Human meniscal cells were also transduced after direct injection of adenoviral vectors (Fig. 1). High levels of gene expression persisted in both lapine and human menisci for twenty weeks in vitro, at which time the experiment was discontinued. In general, injection of the adenovirus vector carrying the lacZ gene into human menisci transduced cells within a wider radius than did injection into lapine menisci.
Indirect Gene Transfer
In Vivo Gene Transfer to Operatively Created Meniscal Lesions
Ex Vivo Gene Transfer with a Fibrin Clot
Ex Vivo Transfer of Transduced Meniscal Cells in Collagen Gel
The results of the present study confirm that, although a meniscal defect becomes filled with fibrous tissue containing fibroblasts by six weeks postoperatively, this tissue is histologically different from normal meniscal tissue. This finding agrees with that of Arnoczky et al.2, who showed that a similarly sized, full-thickness defect in a canine meniscus filled with a fibrin clot was still morphologically different from the adjacent meniscal tissue at three months. Port et al.15 evaluated healing biomechanically after fibrin clots and cultured autogenous marrow cells were placed in operatively created, full-thickness meniscal lesions in goats. They found no enhancement in healing at four months. Their results demonstrated that the fibrin clot, even though it contains chemotactic and mitogenic factors as well as additional cellular components, could not induce complete healing of caprine meniscal lesions. This finding suggests that a single application of the growth factors present in fibrin clots may not promote healing, a circumstance that could reflect the necessity for growth factors to be present within the lesion for an extended period of time. However, with existing technology, high concentrations of growth factors cannot be delivered to sites of meniscal damage in a sustained fashion. In an effort to solve this problem, two of us (P. D. R. and C. H. E.) are developing gene-transfer techniques5 that could be used to promote the sustained, localized production of growth factors within the meniscal lesion in order to improve healing. The present study confirms that gene transfer to meniscal cells is feasible in lapine, canine, and human menisci. With use of the lacZ gene as a marker, we showed that both adenovirally and retrovirally mediated gene transfer can maintain gene expression for at least twenty weeks in vitro. Because the in vitro data suggested that ex vivo gene delivery to a meniscus is more efficient than in vivo gene delivery, the former strategy was employed for gene transfer to operatively created meniscal lesions in experimental animals. Since adenovirus infects nondividing cells, it was possible to utilize this vector in a fibrin-clot technique. The adenoviral vector suspension and the fibrin clot were mixed before implantation into the meniscal lesion. Although few meniscal cells were transduced by this method, cells trapped within the clot expressed the transgene very efficiently and could serve as a local source of growth-factor synthesis. In this sense, the identity of the transduced cells within the clot is not critical, provided that they transduce and express the transgene efficiently. An advantage of the fibrin-clot method is that fibrin clots are already used in operative meniscal repairs, and thus they lend themselves to early use as an adjunct to gene transfer in humans. Moreover, unlike the method involving use of retroviruses, this method does not require cell culture. Adenovirally mediated gene delivery combined with the fibrin-clot technique was successful for delivery of the lacZ gene to the meniscal lesion, with gene expression persisting within the clot for the entire three-week period of the experiment. Retroviruses do not transduce nondividing cells, such as those found in fibrin clots, but they proved to be effective mediators of gene transfer to meniscal cells under in vitro conditions in which the cells divide. Moreover, because retroviruses insert their genetic material stably into the chromosomal DNA of the cells that they infect, there is no dilution of the transferred genes during cell division and it is possible to select for cells expressing the genes of interest. For ex vivo gene delivery with retroviruses, cells were transduced, selected in G418, and embedded in collagen gels before transplantation and insertion into the meniscal defect. This technique was efficient not only in preventing the cells from migrating from the meniscal lesion but also in maintaining the morphology of the transferred meniscal cells. The collagen gel used in this study contained 95 to 98 percent type-I collagen, with the remainder being type-III collagen. Therefore, this collagen-and-cell mixture could be a suitable scaffold for maintaining the integrity of meniscal tissue after transplantation to the lesion. Transgene expression persisted for six weeks after allotransplantation of the retrovirally transduced cells into partial-thickness meniscal defects. The three to six-week duration of gene expression in the meniscal defects noted in the present study may be long enough to enhance healing if a therapeutic gene is utilized. Meniscal cells are known to respond to exogenously supplied growth factors3,9,21, and articular chondrocytes have been shown to respond to transfer of a transforming-growth-factor gene by increasing matrix synthesis1,18. This permits cautious optimism regarding the possibility that these techniques could form the basis of a gene-transfer approach to the repair of meniscal lesions.
NOTE: The authors express sincere thanks to Dr. Rebecca K. Studer, Helga I. Georgescu, Lori Miller, Warren Thompson, Dr. Chang W. Lee, and the Pathology Development Laboratory, Presbyterian-University Hospital.
*One or more of the authors has received or will receive benefits for personal or professional use from a commercial party related directly or indirectly to the subject of this article. Funds were received in total or partial support of the research or clinical study presented in this article. The funding source was Japan Sports Medicine Foundation, Incorporated.
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