The Journal of Bone and Joint Surgery 78:41-8 (1996)
© 1996 The Journal of Bone and Joint Surgery, Inc.
Healing of a Segmental Defect in the Rat Femur with Use of an Extract from a Cultured Human Osteosarcoma Cell-Line (Saos-2). A Preliminary Report*
THOMAS R. HUNT, M.D. ,
JOHN R. SCHWAPPACH, M.D. and
H. CLARKE ANDERSON, M.D. , KANSAS CITY, KANSAS
Investigation performed at the University of Kansas Medical Center, Kansas City
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Abstract
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Devitalized extracts from cultured human osteosarcoma cells (Saos-2) can induce ectopic bone formation. The ability of an extract from Saos-2 cells to stimulate healing of an operatively created four-millimeter defect in the femoral diaphyses of rats was compared with that of collagen and that of autogenous bone graft. Forty adult rats were randomized into four groups of ten each. In Group 1 (controls), no material was placed in the defect; in Group 2, the defect was filled with pure bovine collagen; in Group 3, it was filled with autogenous graft obtained by morseling of the resected segment of the femur; and in Group 4, it was filled with ten milligrams of extract from Saos-2 cells that was mixed with an equal amount of bovine collagen.
Five rats from each group were killed at four weeks and the remaining five, at eight weeks. Each femoral defect was analyzed radiographically and histologically for osseous healing. There was no evidence of healing at either four or eight weeks in Groups 1 and 2. Although there was some new-bone formation in Group 3, none of the defects had united at eight weeks. There was early, almost complete union in all five four-week specimens in Group 4 and complete healing of the defect in four of the five rats assessed at eight weeks. The Saos-2 cell extract was found to be the most effective agent, promoting union by mature lamellar bone within eight weeks.
CLINICAL RELEVANCE: Potential clinical applications of Saos-2-cell bone-inducing extract include the promotion of bone growth in fracture non-unions, in large operatively created defects, and in spinal arthrodeses. It is possible that a bone-inducing extract could enhance osteointegration of porous prosthetic implants.
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Introduction
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Bone grafts have been used for centuries to accelerate bone-healing in operatively created defects26,27. Although autogenous graft from the iliac crest is currently favored for clinical use, demineralized bone matrix also is widely believed to promote osseous healing7,11,20,22. Urist noted that demineralized bone could induce de novo formation of cartilage and bone24. He subsequently succeeded in extracting bone morphogenetic protein from demineralized bone25. Intensive research has led to the isolation of nine separate bone morphogenetic proteins, which have been cloned and sequenced28. These semipurified or purified bone morphogenetic proteins have been used to stimulate healing of operatively created segmental bone defects in animals6,8,10,13,15,16,21,23,29, and clinical trials are beginning in humans. In virtually all of these previous experiments, purified bone morphogenetic protein was implanted along with decalcified bone particles extracted with guanidinium hydrochloride to remove intrinsic bone-inducing activity. The possibility remains that trace amounts of unknown cofactors of bone morphogenetic protein may have been present in the extracted bone matrix. Interaction of these substances with the purified bone morphogenetic proteins may be required to produce osteoinduction4.
Extracts from devitalized cultured human osteosarcoma cells (Saos-2) induce bone formation without the addition of other substances2-4,14. The Saos-2 cell-line (American Type Culture Collection [ATCC], HTB 85), which grows as an apparently matrix-free monolayer with well differentiated epithelial-like features, originated from an osteosarcoma in the lower extremity of an eleven-year-old girl9. It expresses high levels of alkaline phosphatase18 and does not proliferate to form tumors when injected into Nu/Nu mice9. It has been shown19 that Saos-2 cells express measurable levels of messenger RNA (mRNA) for bone morphogenetic protein-1, 2, 3, 4, and 6 and transforming growth factor-ß. The presence of bone morphogenetic protein-1 and 4 and transforming growth factor-ß protein has been confirmed in Saos-2 cells by immunofluorescence, with the presence of transforming growth factor-ß having been confirmed by Western-blot analysis as well7,19. Work is currently under way to identify and quantify other bone morphogenetic proteins in Saos-2 cell extracts and in conditioned media.
The purpose of the current investigation was to evaluate the ability of a semipurified extract of Saos-2 cells to promote osseous union in a long-bone non-union model.
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Materials and Methods
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All operative and experimental procedures were conducted after approval by the Animal Care and Use Committee of the University of Kansas Medical Center. Guidelines set by the National Institutes of Health and the Public Health Service policy on the humane use and care of laboratory animals were strictly followed.
Forty adult male Long Evans rats, ranging in weight from 325 to 375 grams and caged in pairs, were maintained on rodent chow and water ad libitum. They were randomized into four groups of ten rats each: Group 1 (controls), in which no test material was placed in the femoral defect; Group 2, in which the defect was filled with pure bovine collagen (Vitrogen; Collagen Corporation, Palo Alto, California); Group 3, in which the defect was filled with autogenous graft, obtained by morseling the resected segment of the femur; and Group 4, in which the defect was filled with ten milligrams of partially purified bone-inducing extract from Saos-2 cells that was mixed with an equal amount of bovine collagen.
To prepare the partially purified extract, Saos-2 cells were freeze-dried, acetone-defatted, and extracted with four-molar guanidinium hydrochloride at 4 degrees Celsius for forty-eight hours. Residual particles were removed by centrifugation at 3000 revolutions per minute for ten minutes and discarded. The extract containing bone-inducing activity was partially purified by gel filtration on Sephacryl S-200 to obtain a ten to fifty-kilodalton fraction. This gel-filtration fraction was dialyzed exhaustively against phosphate-buffered saline solution at 4 degrees Celsius for twenty-four to forty-eight hours. The resulting dialysate, which contained both solubilized and precipitated proteins, was lyophilized for storage and later resuspended in 3.3 milliliters of a three milligrams per milliliter bovine collagen solution (Vitrogen), frozen, lyophilized, and compressed into sterile number-4 gelatin capsules for implantation. In each group, five rats were killed at four weeks and five, at eight weeks.
All animals had the same operative procedure. A segmental femoral-defect model was modified from that described previously7. The animals were anesthetized with an intraperitoneal injection of a 2:1 mixture of ketamine (100 milligrams per milliliter) and xylazine (twenty milligrams per milliliter), with the rat receiving 0.1 milliliter per 100 grams of body weight. A lateral approach was used to expose the right femoral diaphysis. A five-hole AO/ASIF mini-plate (Synthes, Paoli, Pennsylvania) was positioned on the anterolateral surface of the femur. The proximal two holes and distal two holes in the plate were drilled and tapped for 1.5-millimeter bicortical screws. The most distal hole was filled with a screw. A four-millimeter zone (greater than twice the femoral diameter) was marked on the femur under the middle hole. With use of the distal screw as a pivot, the plate was swung away from the femur and a central diaphyseal defect was created with a water-cooled burr in the previously marked zone (Fig. 1-A). The animals were randomized, and the appropriate implant material was packed into one-half of a sterile number-4 gelatin capsule (Eli Lilly, Indianapolis, Indiana). After irrigation of the medullary canal, the capsule was slid over the distal cut end of the femur (Fig. 1-B). The plate was repositioned and secured with four bicortical screws in the previously prepared holes. The muscle fascia was closed with 3–0 chromic suture, and the skin was closed with 3–0 nylon suture. Unrestricted weight-bearing and activity were allowed as tolerated.
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Figs. 1-A and 1-B: Illustrations of the operative procedure.
Fig. 1-A: Anteroposterior view of the four-millimeter mid-diaphyseal defect that was created in the rat femur with use of a water-cooled burr.
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Lateral view showing the final assembly of the model. One-half of a number-4 gelatin capsule containing the implant material was slid over the cut end of the distal part of the femur. The plate was then secured with two screws at each side of the defect.
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Healing was assessed radiographically immediately postoperatively and at four and eight weeks, with the anesthetized rat positioned prone and the hindlimb externally rotated.
The rats were killed at four or eight weeks postoperatively, the femora were excised and cleaned of all soft tissue, and the specimens were examined grossly and radiographically. The femora were fixed in 10 per cent neutral formalin for at least one week before being processed for histological examination. Coronal histological sections were prepared after decalcification and embedding in paraffin, stained with hematoxylin and eosin, and examined with conventional microscopy. Maps were drawn to approximate the distribution of healing callus, cartilage, reparative bone, regenerated marrow, and fibrous union. These maps were compared with radiographs of each defect to obtain a final impression of the degree of healing achieved with each type of implant that was tested.
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Results
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There was no radiographic evidence of healing, at either four or eight weeks, in Group 1 (the unfilled defects) (Figs. 2-A, 2-B, and 2-C). One screw backed out of each of two plates at four weeks. Histologically, at four and eight weeks all defects were filled with fibrous tissue or skeletal muscle, or both. A very limited amount of new osseous callus was present at the margins of the resection and subperiosteally. The defects were usually bridged by a thin fibrous band at the surface near the stainless-steel plate.
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Figs. 2-A, 2-B, and 2-C: An eight-week specimen in Group 1 (no implant [control]). There was little evidence of healing in this group at four or eight weeks.
Fig. 2-A: Radiograph showing a non-union.
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Diagram showing the defect. A thin layer of reactive new bone, designated osseous callus, was seen capping the edges of the defect and subperiosteally in some areas. (The cross-hatched areas indicate the site of the resection.)
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Group 2 (the defects filled with collagen) also had no radiographic evidence of union at either four or eight weeks (Figs. 3-A, 3-B, and 3-C). Radiographs revealed a small quantity of callus near the margins of the resection (Fig. 3-A). There was one bent plate at eight weeks. Histologically, residual collagen was present within the defects even at eight weeks. The resected ends of the bone were capped by new bone, and there was subperiosteal new bone. Thin strands of fibrous tissue connected the cut ends except where they had been impeded by residual collagen.
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Figs. 3-A, 3-B, and 3-C: An eight-week specimen in Group 2 (collagen implant).
Fig. 3-A: Radiograph showing a non-union.
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Diagram showing the non-union. As at four weeks, an unresorbed collagen plug filled the defect, which was united only by a thin band of fibrous tissue. (The cross-hatched areas indicate the site of the resection; M = marrow and B = bone.)
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In Group 3 (the defects filled with autogenous graft), there was no radiographic sign of union at four or eight weeks (Figs. 4-A, 4-B, and 4-C). There was evidence of resorption of the graft, which continued until eight weeks, but small particles of autogenous graft were still present at eight weeks (Figs. 4-B and 4-C). Two plates were bent at eight weeks. Grossly, there was firm fibrous material in the defects at four and eight weeks. Histologically, none of the defects were spanned completely by either cartilage or new bone. At four weeks, there were relatively more dead particles of autogenous graft in the defects as well as active formation of callus at the cut ends of the femur. By eight weeks, most of the dead particles had been resorbed, with large projections of new bone extending from the cut ends of the femur that did not unite the osseous segments.
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Figs. 4-A, 4-B, and 4-C: An eight-week specimen in Group 3 (autogenous graft).
Fig. 4-A: Radiograph showing a non-union. Delicate projections of osseous callus can be seen extending into the defect from the cut edge of the bone and periosteum.
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Diagram showing the defect. Bone repair was more advanced at this time-period in this group, with projections of osseous callus sometimes extending almost across the defect. The cut edges were also capped by callus. Small pieces of unresorbed, dead autogenous graft were present but appeared to be smaller and fewer than at four weeks. (The cross-hatched areas indicate the site of the resection; M = marrow.)
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Photomicrograph. The non-union is approaching union, as was the case with all of the animals in Group 3 (hematoxylin and eosin, x 20).
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In Group 4 (the defects filled with bone-inducing extract), there was histological evidence of early, almost complete osseous union in all five animals assessed at four weeks. Complete radiographic union was present at eight weeks in four of the five rats examined at that time (Figs. 5-A, 5-B, and 5-C). The defect in the fifth rat was approximately 80 per cent united. Careful review of the histological specimens from this rat revealed evidence of bone-cell necrosis at one surface of the osteotomy, presumably caused by thermal necrosis from the burr, which inhibited healing at that particular surface in this rat. There were two bent plates at four weeks. Grossly, there was hard material in the defects, conferring stability to the femur, at both four and eight weeks. Histologically, cartilage and new bone filled approximately 95 per cent of each defect by four weeks. By eight weeks, a new outer condensation of lamellar bone resembling a cortex had appeared, encasing a re-formed medullary canal (Figs. 5-B and 5-C). There was histological evidence of bone-remodeling, with osteoblastic activity continuing at the ends of the bone and active resorption and formation occurring subperiosteally. New bone spanned the entire defect in all animals. One edge of the osteotomy had not fused with osseous callus, presumably because of thermal damage to the bone cells situated at that edge.
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Figs. 5-A, 5-B, and 5-C: An eight-week specimen in Group 4 (bone-inducing extract and collagen).
Fig. 5-A: Radiograph showing complete osseous union, which occurred in four of the five rats in this group.
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Diagram of the defect. Complete union was achieved by mature osseous callus bridging 100 per cent of the defect. Lamellar bone at the cortex is seen encompassing a re-formed medullary canal, complete with trabecular bone and marrow. (The cross-hatched areas indicate the site of the resection.)
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Discussion
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A human osteosarcoma cell-line (Saos-2) has been maintained in perpetual cell culture since its initial isolation and cultivation in 19759. Recently, Anderson et al. discovered that Saos-2 cells are osteoinductive, whether they are implanted alone (as freeze-dried cells) or as a guanidinium-hydrochloride extract3. This bone-inducing activity is unique among the several available human and animal osteosarcoma cell-lines that have been tested2,3. On the basis of these observations, we examined the effect of Saos-2 cell extracts on the healing of segmental bone defects, and the results were impressive: the Saos-2 extracts elicited healing that was both faster and better than that obtained with autogenous bone grafts.
In preliminary experiments, we observed that unpurified Saos-2 cell extracts that were osteoinductive in Nu/Nu mice generated a marked inflammatory response and did not induce heterotopic bone formation when they were implanted into immunocompetent Long Evans rats. We reasoned that the inflammation seen in outbred rats was the result of their becoming sensitized to Saos-2 cell proteins, which are of human origin. We attempted to isolate a fraction of Saos-2 cell proteins that would not cause inflammation in rats. The partially purified extracts used in this study, consisting only of proteins with molecular weights in the range of ten to fifty kilodaltons, did not generate an inflammatory response when they were implanted intramuscularly in Long Evans rats, and they contained all of the factors necessary for osteoinduction.
The osteoinductive activity residing in extracts of Saos-2 cells may not be due to a single morphogen or cytokine but, rather, to a combination of bone-growth and morphogenetic factors. In a recent study19, evidence indicated that Saos-2 cells express mRNAs for bone morphogenetic protein-1, 2, 3, 4, and 6 and transforming growth factor-ß. Also, immunofluorescence demonstrated bone morphogenetic protein-1 and 4 and transforming growth factor-ß proteins in the cytoplasm of Saos-2 cells, with the presence of transforming growth factor-ß shown by Western-blot analysis as well17,19. Initially, the data suggested that any one of these bone morphogenetic proteins or transforming growth factor-ß might be the only factor required for bone induction. However, in parallel experiments, we analyzed the pattern of expression of bone morphogenetic proteins in an alternative human osteosarcoma cell-line, U2OS, which has consistently failed to induce bone formation in Nu/Nu mice. To our surprise, the non-osteoinductive U2OS cells expressed relatively high levels of mRNAs for bone morphogenetic protein-2 through 7 and transforming growth factor-ß4,19. In fact, U2OS cells exceeded Saos-2 cells in expressing bone morphogenetic protein-2, 4, 5, 6, and 7. U2OS cells may be non-osteoinductive because, although they express appropriate bone-growth and morphogenic factors, they do so in inadequate relative concentrations. It is noteworthy that studies of pure or recombinant bone morphogenetic proteins have suggested that multiple bone morphogenetic proteins working together may be more effective in inducing bone than a single factor working alone5,12. On the basis of these observations, our current working hypothesis is that Saos-2 cells contain an optimum admixture of bone-growth factors, both known and perhaps unknown, that are sufficient to induce bone formation without the addition of undefined factors.
In the experiments reported here, chemically pure non-osteoinductive bovine collagen was added to the partially purified Saos-2 cell extracts in order to retain bone-inducing activity at the site of bone induction long enough to effect an interaction with the host's osteoprogenitor cells. Thus, Saos-2 cell extracts that require only a pure collagen carrier to support activity may be preferable to recombinant single bone morphogenetic proteins that require a carrier composed of guanidinium-hydrochloride-extracted decalcified bone particles to be active in vivo6,10,12,15,16,21,28. Such decalcified bone particles, while not able to induce bone alone, clearly contain many undefined protein components that might interact with and enhance the osteoinductivity of a pure recombinant bone morphogenetic protein.
In summary, a semipurified, low-molecular-weight extract of Saos-2 cells combined solely with a collagen carrier effectively induced healing in a diaphyseal defect non-union model in the rat femur. The extract contained potent bone-inducing activity and was more effective than the use of no implant, collagen, or autogenous graft. There was no evidence of rejection or neoplasia. Purified Saos-2 cell components may be an alternative source of bone morphogenetic proteins and other growth factors, which might be used instead of recombinant bone morphogenetic proteins to elicit the repair of segmental bone defects.
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Footnotes
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*No benefits in any form have been received or will be received 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 National Institutes of Health Grant DEO 5262 from the American Fracture Association and the Mid-America Orthopaedic Association.
Department of Orthopaedic Surgery, Virginia Mason Medical Center, Seattle, Washington 98111.
Department of Orthopaedic Surgery, University of Colorado Medical Center, 4701 East 9th Avenue, Denver, Colorado 80262.
Department of Pathology and Laboratory Medicine, University of Kansas Medical Center, 3901 Rainbow Boulevard, Kansas City, Kansas 66160-7410.
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Abstract
Introduction
