- Email: [email protected]
Tissue-engineered composites of bone and cartilage for mandible condylar reconstruction
J Oral Maxillofac Surg 59:185-190, 2001
Tissue-Engineered Composites of Bone and Cartilage for Mandible Condylar Reconstruction Yulai Weng, DDS, BS,* Yilin Cao, MD, PhD,† Carlos Arevalo Silva, MD,‡ Martin P. Vacanti, MD,§ and Charles A. Vacanti, MD㛳 Purpose:
This study evaluated the feasibility of creating a tissue-engineered adult human mandible condyle composite of bone and cartilage. Materials and Methods: A polymer template composed of polyglycolic acid (PGA) and polylactic acid (PLA), and formed in the shape of the human mandible condyle, was seeded with osteoblasts isolated from a bovine periosteum suspended in calcium alginate. Chondrocytes isolated from the same calf suspended in 30% pluronic were then “painted” onto the articular surface of the scaffold, and it was then implanted into subcutaneous pockets on the dorsum of athymic mice. Animals were divided into 3 groups: group I (n ⫽ 6) received a PGA/PLA scaffold saturated with hydrogels not containing cells; group II (n ⫽ 6) received scaffolds seeded with both cell types suspended in saline rather than hydrogels; and group III (n ⫽ 6) received scaffolds seeded with both cell types suspended in hydrogel composites. Constructs were harvested after 12 weeks and evaluated grossly and microscopically by using histologic stains. Results: In group I, the constructs formed a small mass without evidence of new bone or cartilage. In group II, the constructs were small and irregular. Microscopically they contained scattered islands of bone and cartilage. All specimens in group III retained their original condylar shape and were quite firm. Microscopic evaluation indicated trabecular bone interfacing with hyaline cartilage on the articulating surface. Conclusion: These findings show that the composites of bone and cartilage can be engineered to serve as condylar substitutes. The interdigitation of bone and cartilage at their interface is similar to the normal interface of these composite tissues seen in articulating joints. © 2001 American Association of Oral and Maxillofacial Surgeons Severe destruction of the condylar process of the temporomandibular joint (TMJ) resulting from tumors, trauma, and ankylosis remains a challenge for oral and maxillofacial reconstructive surgery. Loss of TMJ function may result in alterations in speech, swallowing, mastication, mandibular growth, and
facial symmetry. The ideal goal would be to reconstruct a mandibular condyle that is similar to the original, containing an articular surface of cartilage and subchondral bone. Modern techniques to address these issues depend on the use of autologous bone and cartilage, tissue allografts, or prosthe-
*Research Fellow, Center for Tissue Engineering, Department of Anesthesiology, University of Massachusetts Medical School, Worcester, MA; and Research Fellow, Center for Tissue Engineering, Shanghai Second Medical University, Shanghai, People’s Republic of China. †Research Associate Professor, Center for Tissue Engineering, Department of Anesthesiology, University of Massachusetts Medical School, Worcester, MA; and Professor of Surgery, Center for Tissue Engineering, Shanghai Second Medical University, Shanghai, People’s Republic of China. ‡Research Fellow, Center for Tissue Engineering, Department of Anesthesiology, University of Massachusetts Medical School, Worcester, MA.
§Research Assistant Professor, Center for Tissue Engineering and Department of Anesthesiology; Assistant Professor of Pathology, Department of Pathology, University of Massachusetts Medical School, Worcester, MA. 㛳Professor and Chair, Center for Tissue Engineering, Department of Anesthesiology, University of Massachusetts Medical School, Worcester, MA. Address correspondence and reprint requests to Dr Vacanti: Professor and Chair, Center for Tissue Engineering, Department of Anesthesiology, University of Massachusetts Medical School, 55 Lake Ave North, Worcester, MA 01655; e-mail: [email protected]. © 2001 American Association of Oral and Maxillofacial Surgeons
0278-2391/01/5902-0009$35.00/0 doi:10.1053/joms.2001.20491
185
186 ses.1-4 Each of these approaches have specific complications. Autologous grafts, such as rib, metatarsal, sternoclavicular joint, and cranial bone have been used for decades.3-10 Unfortunately, their supply is limited, they suffer from donor site morbidity, and they are occasionally not suitable for the expected reconstruction owing to poor tissue quality or the difficult sculpting needed. Recently, a report concluded that there were major complications associated with the implantation of autologous grafts in the treatment of TMJ ankylosis.11 Allografts also are limited in supply and often trigger immunologic rejection. Although use of alloplastic implants may decrease operating time,4,12 these implants are associated with an increased susceptibility to infection and have an uncertain long-term durability, with occasional extrusion from the operative site. In addition, alloplastic materials often fail to meet the dynamic physiologic needs of the TMJ. Thus, establishment of normal occlusion and TMJ contour is uncertain. To overcome these difficulties, a technology referred to as tissue engineering has evolved,13-19 which involves the morphogenesis of new tissue from dissociated cells and biodegradable polymers. Hence, tissue engineering offers the potential to grow a composite of bone and cartilage in precisely predetermined shapes.20 To our knowledge, the use of tissue engineering to form composites of bone and cartilage for human mandibular condyle reconstruction has not been reported in the literature. The primary purpose of this study was to evaluate the feasibility of creating tissueengineered composites of bone and cartilage for surgical replacement of the adult human mandibular condyle.
TISSUE-ENGINEERED MANDIBULAR CONDYLE OSTEOBLAST CULTURE AND HARVEST
Osteoblasts were isolated from a bovine periosteum-derived explant culture system.15,17 The periosteum was harvested from a freshly slaughtered (⬍6 hours) calf forelimb and cut into 1-cm2 pieces under sterile conditions. Minced periosteum was placed into 100-mm tissue culture dishes in the presence of 10 mL Medium 199 (Gibco, Grand Island, NY), supplemented with 10% fetal calf serum, ascorbic acid (50 mg/mL), L-glutamine (292 mg/mL), penicillin (100 U/mL), and streptomycin (100 mg), and allowed to incubate at 37°C in a 5% CO2 environment. Culture media were changed every 3 or 4 days until a confluent monolayer of cells was observed on phase-contrast microscopy. Once a confluent monolayer was detected, the periosteal specimen was removed, and osteoblasts were harvested by digestion with 0.05% Trypsin-ethylenediaminetetra-acetic acid. Cell number and viability were determined by using a hemocytometer and trypan blue vital dye exclusion. Cells that migrated from the periosteal explant after 3 weeks were stained with an alkaline phosphatase reaction consisting of 0.05% Naphthol AS-MX (Sigma, St Louis, MO), N,N dimethylformamide (Sigma), 0.1% Fast Red TR salt (Sigma), and 0.2 mol/L Tris buffer at a pH of 9.0. OSTEOBLAST-CALCIUM ALGINATE PREPARATION
Isolated periosteal cells were resuspended in a 1.5% sterile sodium alginate (Protan, Portsmouth, NH) solution (0.1 mol/L K2HOP4, 0.135 mol/L NaCl, pH 7.4), which had previously been sterilized by autoclaving, to yield a cellular concentration of 5 ⫻ 107/mL in a 1.5% alginic acid solution and stored at 4°C until use. Before seeding onto the scaffold, 0.2 g sterilized
Materials and Methods CONSTRUCTION OF POLYMER DEVICES
Using an adult human TMJ condylar process as a model, synthetic biodegradable polymer constructs were created. A synthetic nonwoven mesh of polyglycolic acid fibers, with a diameter of 15 m interfiber and spaces averaging 100 to 200 m, was immersed for approximately 2 seconds in a 1.5% (wt/ vol) solution of polylactic acid in methylene chloride. After immersion, the fabric was removed, shaped into the form of an adult human TMJ condylar process, and allowed to dry. The polymer devices were placed in 35-mm polystyrene tissue culture dishes (Costar, Cambridge, MA), gas (ethylene oxide) sterilized overnight, and set aside.
FIGURE 1. Left: The mold of a human mandible condyle. Middle: Scaffold is made from PGA/PLA construct before implantation. Right: A specimen from group III, in which the calcium alginate-osteoblasts and pluronic-chondrocytes were applied at the same time, displays a morphology nearly identical to that of the initial implant and mimics the complex configuration of the original adult human mandibular condyle.
WENG ET AL
CaSO4 powder was added to each milliliter of the osteoblast-alginic acid mixture to initiate gel formation. ISOLATION OF CHONDROCYTES
The forelimbs used for harvesting of periosteum were dissected under sterile conditions to expose the articular surfaces of the glenohumeral and humeroulnar joints. Cartilage fragments were curetted off the articular surface of each joint and placed in Dulbecco’s phosphate-buffered saline (PBS) (Gibco). Using a method similar to that described by Klagsbrun,21 the
FIGURE 2. Tissue-engineered mandibular condyle. A, Photomicrograph showing fragments of cartilage, trabecular bone, and the interface of cartilage and bone (Hematoxylin and eosin, original magnification ⫻100). B, Higher-magnification view clearly shows that the cartilage has three layers: a superficial zone, a transitional zone, and a deep radial zone with columnar organization of the chondrocytes. Abutting the cartilage is trabecular bone containing osteoblasts. Chondrocytes infiltrate the underlying osseous substrate, forming an interdigitated cartilaginous-osseous interface (original magnification ⫻200).
187 fragments were subjected to collagenase II (3 mg/mL) (Worthington Biochemical Corp, Freehold, NJ) digestion at 37°C for 12 to 18 hours. The resulting cell suspension was passed through a sterile 250-m polypropylene mesh filter (Spectra/Mesh 146-426; Spectrum Medical Industries, Inc, Laguna Hills, CA). The filtrate was centrifuged at 6,000 rpm and the resulting cell pellet was washed twice with copious amounts of PBS without Ca2⫹. The cell number was calculated by using a hemocytometer, and cell viability was determined by using trypan blue dye exclusion (Sigma-Aldrich, Irvine, CA).
188
TISSUE-ENGINEERED MANDIBULAR CONDYLE
FIGURE 3. Photomicrograph showing an intense, uniform orange-red staining of the cartilage matrix, indicating proteoglycan production. (Safranin-O, original magnification ⫻200.)
CHONDROCYTE-PLURONIC F127 PREPARATION
An aliquot of the chondrocyte suspension was mixed with a 30% (wt/vol) solution of co-polymer hydrogel of pluronic F127 (ethylene and propylene oxide) (BASE, Mount Olive, NJ) at a cellular concentration of 5 ⫻ 107 cells/mL. Aliquots containing 300 L of this mixture were painted onto the articular surfaces of the scaffolds that had been seeded with periosteal cells in alginate. SPECIMEN IMPLANTATION
Under general anesthesia (methoxyflurane; Pittmann and Moores, Mundelein, IL), and using sterile
FIGURE 4. Photomicrograph shows an intense blue-green color, indicating collagen present in both the bone and cartilage matrix. (Masson’s trichrome; original magnification ⫻200).
surgical techniques, athymic male mice (nu/nu) (MGH, Boston, MA) 4 to 6 weeks of age each received 1 polymer construct in a dorsal subcutaneous pockets according to a random group assignment. One control group (group I) and 2 experimental groups (groups II and III) were studied. The control group (n ⫽ 6) consisted of animals receiving scaffolds saturated with calcium alginate and pluronic F127 alone, but not containing cells. Group II (n ⫽ 6) consisted of animals receiving scaffolds seeded with osteoblasts and chondrocytes in saline without the hydrogels calcium alginate and pluronic F127. Group III (n ⫽ 6) consisted of animals receiving scaffolds saturated
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with the hydrogels alginate and pluronic F127 seeded with the 2 cell types, as previously described. SAMPLE HARVESTING
The animals were killed 12 weeks after implantation with an overdose of inhalation anesthesia. Specimens were dissected free of surrounding soft tissue and fixed in 10% phosphate-buffered formalin (Fisher Scientific, Pittsburgh, PA) for gross and histologic analysis. HISTOLOGIC ANALYSIS
After fixation in 10% phosphate-buffered formalin for 24 hours, the specimens were embedded in paraffin and sectioned. Using standard histologic techniques, serial sections were stained with hematoxylin and eosin, Safranin-O, Masson’s trichrome, and Von Kossa’s stain.
Results None of the implants showed extrusion or infection. Group I (control group) specimens showed no evidence of bone and cartilage formation. Groups II and III differed significantly in the overall 3-dimensional appearance of the explants. Specimens from group II showed only rudimentary 3-dimensional structures. These specimens were smaller and had an irregular shape when compared with specimens obtained from group III. Specimens excised from group III displayed a morphology nearly identical to that of the initial implant and mimicked the complex configuration of the original adult human TMJ condylar process (Fig 1). Specimens in both groups II and III showed evidence of new bone and cartilage formation on gross examination. Histologic examination using a standard hematoxylin and eosin (H&E) stain also showed bone and cartilage formation in these groups. In group III, a 2- to 3-mm coating of firm, white, opaque cartilage was noted on the articulating surface, which was adherent to the underlying bone. Microscopic evaluation with H&E and Safranin-O stains showed hyaline cartilage closely resembling native articular cartilage. Several sections showed the hyaline cartilage to be organized in 3 distinct layers: a superficial zone, a transitional zone, and an inferior radial zone with columnar organization of the chondrocytes. The cartilage was also organized into lobules with round, angular lacunae each containing a single chondrocyte. Abutting the cartilage was trabecular bone lined with osteoblasts (Fig 2). Microscopic evaluation using polarized light showed organized trabecular bone, with many areas showing organized concentric lamellae. The Von Kossa stain showed mineralization as the cartilage abutted
the bone. Chondrocytes could be seen infiltrating the underlying osseous substrate, forming a cartilaginousosseous interface. Ossification was located primarily in the center of the mature bone in which collagen bundles organized into bone lamella. Osteocytes in their lacunae were surrounded by bone matrix. Safranin-O stains of tissue sections from the group III specimens showed purple and green staining of bone/cartilage consistent with the presence of proteoglycan and glycosaminoglycans, such as chondroitin sulfate (Fig 3). Masson’s trichrome stain in group III showed the presence of collagen and suggested the presence of type I and II collagen, respectively, for bone and cartilage (Fig 4). Occasional remnants of polymer were seen in some areas.
Discussion This study shows that osteoblasts and chondrocytes can form in in vivo composites of bone and cartilage that may be suitable for condylar reconstruction. Only implants seeded with cells in hydrogel replicated the complex configuration of the human mandibular condyle. This suggests that the hydrogels may improve the potential of the scaffolds to maintain a specific shape. This study showed that tissues can be successfully engineered to generate composites of bone/cartilage for condyle replacement by using a combination of synthetic scaffolds into which cells are seeded at appropriate concentrations. Future studies are needed to investigate the physical properties in a controlled manner using biomechanical and dynamic force protocols. Articular chondrocytes, rather than cells from fibrocartilage, were used to avoid the rapid overgrowth of fibroblasts in tissue culture associated with the use of chondrocytes from fibrocartilage. Articular cartilage contains only chondrocytes and thus avoids fibroblast overgrowth.15 Of great interest is that the fabricated framework was of a clinically suitable size. This demonstrates that sufficient tissue can be generated when properly engineered.
References 1. Lindqvist C, Jokinen J, Paukku P, et al: Adaptation of autogenous costochondral grafts used for temporomandibular joint reconstruction. J Oral Maxillofac Surg 46:465, 1988 2. Nelson CL, Buttrum JD: Costochondral grafting for posttraumatic temporomandibular joint reconstruction: A review of six cases. J Oral Maxillofac Surg 47:1030, 1989 3. Behnia H, Motamedi MHK, Tehranchi A: Use of activator appliances in pediatric patients treated with costochontral grafts for temporomandibular joint ankylosis: Analysis of 13 cases. J Oral Maxillofac Surg 55:1408, 1997 4. Marx RE: Mandibular reconstruction. J Oral Maxillofac Surg 51:466, 1993 5. Daniels S, Ellis E, Carlson DS: Histologic analyses of costochondral and sternoclavicular grafts in the TMJ of the juvenile monkey. J Oral Maxillofac Surg 45:675, 1987
190 6. Dodson TB, Bays RA, Pfeffle RC, et al: Cranial bone graft to reconstruct the mandibular condylar in Macaca mulatta. J Oral Maxillofac Surg 55:260, 1997 7. Carlson ER, Marx R: Mandibular reconstruction using cancellous cellular bone grafts. J Oral Maxillofac Surg 54:889, 1996 8. Hinton RJ, Stinson JL: Effect of postoperative diet on condylar cartilage response to discectomy. J Oral Maxillofac Surg 55: 1259, 1997 9. Hall HD, Werther JR: Results of reoperation after failed modified condylotomy. J Oral Maxillofac Surg 55:1250, 1997 10. Tidstrom KD, Keller EE: Reconstruction of mandible discontinuity with autogenous iliac bone graft: Report of 34 consecutive patients. J Oral Surg 48:336, 1990 11. Kinoshita Y, Kobayashi M, Hidaka T, et al: Reconstruction of mandibular continuity defects in dogs using poly(L-Lactide) mesh and autogenic particulate cancellous bone and marrow: Preliminary report. J Oral Maxillofac Surg 55:718, 1997 12. Kent JN, Misiek DJ, Akin RK, et al: Temporomandibular joint condylar prothesis: A ten year report. J Oral Surg 41:245, 1983 13. Vacanti JP, Morse MA, Saltzman WM, et al: Selective cell transplantation using bioabsorbable artificial polymers as matrices. J Pediatr Surg 23:3, 1988
TISSUE-ENGINEERED MANDIBULAR CONDYLE 14. Vacanti JP: Beyond transplantation. Arch Surg 123:545, 1988 15. Cao Y, Vacanti JP, Paige KT, et al: Transplantation of chondrocytes utilizing a polymer-cell construct to produce tissue-engineered cartilage in the shape of a human ear. Plast Reconstr Surg 100:297, 1997 16. Cao Y, Vacanti JP, Ma X, et al: Generation of neo-tendon using synthetic polymers seeded with tenocytes. Transplant Proc 26:3390, 1994 17. Vacanti CA, Langer R, Schloo B, et al: Synthetic polymer seeded with chondrocytes provide a template for new cartilage formation. Plast Reconstr Surg 88:753, 1991 18. Vacanti CA, Kim W, Upton J, et al: The efficacy of periosteal cells compared with chondrocytes in the tissue engineered repair of bone defects. Tissue Eng 1:301, 1995 19. Vacanti CA, Upton J: Tissue-engineered morphogenesis of cartilage and bone by means of cell transplantation using synthetic. Clin Plast Surg 21:445, 1994 20. Puelacher WG, Wisser J, Vacanti CA, et al: Temporomandibular joint disc replacement made by tissue-engineered growth of cartilage. J Oral Maxillofac Surg 52:1172, 1994 21. Klagsbrun M: Large-scale preparation of chondrocytes. Methods Enzymol 58:560, 1979
Tissue-Engineered Composites of Bone and Cartilage for Mandible Condylar Reconstruction Yulai Weng, DDS, BS,* Yilin Cao, MD, PhD,† Carlos Arevalo Silva, MD,‡ Martin P. Vacanti, MD,§ and Charles A. Vacanti, MD㛳 Purpose:
This study evaluated the feasibility of creating a tissue-engineered adult human mandible condyle composite of bone and cartilage. Materials and Methods: A polymer template composed of polyglycolic acid (PGA) and polylactic acid (PLA), and formed in the shape of the human mandible condyle, was seeded with osteoblasts isolated from a bovine periosteum suspended in calcium alginate. Chondrocytes isolated from the same calf suspended in 30% pluronic were then “painted” onto the articular surface of the scaffold, and it was then implanted into subcutaneous pockets on the dorsum of athymic mice. Animals were divided into 3 groups: group I (n ⫽ 6) received a PGA/PLA scaffold saturated with hydrogels not containing cells; group II (n ⫽ 6) received scaffolds seeded with both cell types suspended in saline rather than hydrogels; and group III (n ⫽ 6) received scaffolds seeded with both cell types suspended in hydrogel composites. Constructs were harvested after 12 weeks and evaluated grossly and microscopically by using histologic stains. Results: In group I, the constructs formed a small mass without evidence of new bone or cartilage. In group II, the constructs were small and irregular. Microscopically they contained scattered islands of bone and cartilage. All specimens in group III retained their original condylar shape and were quite firm. Microscopic evaluation indicated trabecular bone interfacing with hyaline cartilage on the articulating surface. Conclusion: These findings show that the composites of bone and cartilage can be engineered to serve as condylar substitutes. The interdigitation of bone and cartilage at their interface is similar to the normal interface of these composite tissues seen in articulating joints. © 2001 American Association of Oral and Maxillofacial Surgeons Severe destruction of the condylar process of the temporomandibular joint (TMJ) resulting from tumors, trauma, and ankylosis remains a challenge for oral and maxillofacial reconstructive surgery. Loss of TMJ function may result in alterations in speech, swallowing, mastication, mandibular growth, and
facial symmetry. The ideal goal would be to reconstruct a mandibular condyle that is similar to the original, containing an articular surface of cartilage and subchondral bone. Modern techniques to address these issues depend on the use of autologous bone and cartilage, tissue allografts, or prosthe-
*Research Fellow, Center for Tissue Engineering, Department of Anesthesiology, University of Massachusetts Medical School, Worcester, MA; and Research Fellow, Center for Tissue Engineering, Shanghai Second Medical University, Shanghai, People’s Republic of China. †Research Associate Professor, Center for Tissue Engineering, Department of Anesthesiology, University of Massachusetts Medical School, Worcester, MA; and Professor of Surgery, Center for Tissue Engineering, Shanghai Second Medical University, Shanghai, People’s Republic of China. ‡Research Fellow, Center for Tissue Engineering, Department of Anesthesiology, University of Massachusetts Medical School, Worcester, MA.
§Research Assistant Professor, Center for Tissue Engineering and Department of Anesthesiology; Assistant Professor of Pathology, Department of Pathology, University of Massachusetts Medical School, Worcester, MA. 㛳Professor and Chair, Center for Tissue Engineering, Department of Anesthesiology, University of Massachusetts Medical School, Worcester, MA. Address correspondence and reprint requests to Dr Vacanti: Professor and Chair, Center for Tissue Engineering, Department of Anesthesiology, University of Massachusetts Medical School, 55 Lake Ave North, Worcester, MA 01655; e-mail: [email protected]. © 2001 American Association of Oral and Maxillofacial Surgeons
0278-2391/01/5902-0009$35.00/0 doi:10.1053/joms.2001.20491
185
186 ses.1-4 Each of these approaches have specific complications. Autologous grafts, such as rib, metatarsal, sternoclavicular joint, and cranial bone have been used for decades.3-10 Unfortunately, their supply is limited, they suffer from donor site morbidity, and they are occasionally not suitable for the expected reconstruction owing to poor tissue quality or the difficult sculpting needed. Recently, a report concluded that there were major complications associated with the implantation of autologous grafts in the treatment of TMJ ankylosis.11 Allografts also are limited in supply and often trigger immunologic rejection. Although use of alloplastic implants may decrease operating time,4,12 these implants are associated with an increased susceptibility to infection and have an uncertain long-term durability, with occasional extrusion from the operative site. In addition, alloplastic materials often fail to meet the dynamic physiologic needs of the TMJ. Thus, establishment of normal occlusion and TMJ contour is uncertain. To overcome these difficulties, a technology referred to as tissue engineering has evolved,13-19 which involves the morphogenesis of new tissue from dissociated cells and biodegradable polymers. Hence, tissue engineering offers the potential to grow a composite of bone and cartilage in precisely predetermined shapes.20 To our knowledge, the use of tissue engineering to form composites of bone and cartilage for human mandibular condyle reconstruction has not been reported in the literature. The primary purpose of this study was to evaluate the feasibility of creating tissueengineered composites of bone and cartilage for surgical replacement of the adult human mandibular condyle.
TISSUE-ENGINEERED MANDIBULAR CONDYLE OSTEOBLAST CULTURE AND HARVEST
Osteoblasts were isolated from a bovine periosteum-derived explant culture system.15,17 The periosteum was harvested from a freshly slaughtered (⬍6 hours) calf forelimb and cut into 1-cm2 pieces under sterile conditions. Minced periosteum was placed into 100-mm tissue culture dishes in the presence of 10 mL Medium 199 (Gibco, Grand Island, NY), supplemented with 10% fetal calf serum, ascorbic acid (50 mg/mL), L-glutamine (292 mg/mL), penicillin (100 U/mL), and streptomycin (100 mg), and allowed to incubate at 37°C in a 5% CO2 environment. Culture media were changed every 3 or 4 days until a confluent monolayer of cells was observed on phase-contrast microscopy. Once a confluent monolayer was detected, the periosteal specimen was removed, and osteoblasts were harvested by digestion with 0.05% Trypsin-ethylenediaminetetra-acetic acid. Cell number and viability were determined by using a hemocytometer and trypan blue vital dye exclusion. Cells that migrated from the periosteal explant after 3 weeks were stained with an alkaline phosphatase reaction consisting of 0.05% Naphthol AS-MX (Sigma, St Louis, MO), N,N dimethylformamide (Sigma), 0.1% Fast Red TR salt (Sigma), and 0.2 mol/L Tris buffer at a pH of 9.0. OSTEOBLAST-CALCIUM ALGINATE PREPARATION
Isolated periosteal cells were resuspended in a 1.5% sterile sodium alginate (Protan, Portsmouth, NH) solution (0.1 mol/L K2HOP4, 0.135 mol/L NaCl, pH 7.4), which had previously been sterilized by autoclaving, to yield a cellular concentration of 5 ⫻ 107/mL in a 1.5% alginic acid solution and stored at 4°C until use. Before seeding onto the scaffold, 0.2 g sterilized
Materials and Methods CONSTRUCTION OF POLYMER DEVICES
Using an adult human TMJ condylar process as a model, synthetic biodegradable polymer constructs were created. A synthetic nonwoven mesh of polyglycolic acid fibers, with a diameter of 15 m interfiber and spaces averaging 100 to 200 m, was immersed for approximately 2 seconds in a 1.5% (wt/ vol) solution of polylactic acid in methylene chloride. After immersion, the fabric was removed, shaped into the form of an adult human TMJ condylar process, and allowed to dry. The polymer devices were placed in 35-mm polystyrene tissue culture dishes (Costar, Cambridge, MA), gas (ethylene oxide) sterilized overnight, and set aside.
FIGURE 1. Left: The mold of a human mandible condyle. Middle: Scaffold is made from PGA/PLA construct before implantation. Right: A specimen from group III, in which the calcium alginate-osteoblasts and pluronic-chondrocytes were applied at the same time, displays a morphology nearly identical to that of the initial implant and mimics the complex configuration of the original adult human mandibular condyle.
WENG ET AL
CaSO4 powder was added to each milliliter of the osteoblast-alginic acid mixture to initiate gel formation. ISOLATION OF CHONDROCYTES
The forelimbs used for harvesting of periosteum were dissected under sterile conditions to expose the articular surfaces of the glenohumeral and humeroulnar joints. Cartilage fragments were curetted off the articular surface of each joint and placed in Dulbecco’s phosphate-buffered saline (PBS) (Gibco). Using a method similar to that described by Klagsbrun,21 the
FIGURE 2. Tissue-engineered mandibular condyle. A, Photomicrograph showing fragments of cartilage, trabecular bone, and the interface of cartilage and bone (Hematoxylin and eosin, original magnification ⫻100). B, Higher-magnification view clearly shows that the cartilage has three layers: a superficial zone, a transitional zone, and a deep radial zone with columnar organization of the chondrocytes. Abutting the cartilage is trabecular bone containing osteoblasts. Chondrocytes infiltrate the underlying osseous substrate, forming an interdigitated cartilaginous-osseous interface (original magnification ⫻200).
187 fragments were subjected to collagenase II (3 mg/mL) (Worthington Biochemical Corp, Freehold, NJ) digestion at 37°C for 12 to 18 hours. The resulting cell suspension was passed through a sterile 250-m polypropylene mesh filter (Spectra/Mesh 146-426; Spectrum Medical Industries, Inc, Laguna Hills, CA). The filtrate was centrifuged at 6,000 rpm and the resulting cell pellet was washed twice with copious amounts of PBS without Ca2⫹. The cell number was calculated by using a hemocytometer, and cell viability was determined by using trypan blue dye exclusion (Sigma-Aldrich, Irvine, CA).
188
TISSUE-ENGINEERED MANDIBULAR CONDYLE
FIGURE 3. Photomicrograph showing an intense, uniform orange-red staining of the cartilage matrix, indicating proteoglycan production. (Safranin-O, original magnification ⫻200.)
CHONDROCYTE-PLURONIC F127 PREPARATION
An aliquot of the chondrocyte suspension was mixed with a 30% (wt/vol) solution of co-polymer hydrogel of pluronic F127 (ethylene and propylene oxide) (BASE, Mount Olive, NJ) at a cellular concentration of 5 ⫻ 107 cells/mL. Aliquots containing 300 L of this mixture were painted onto the articular surfaces of the scaffolds that had been seeded with periosteal cells in alginate. SPECIMEN IMPLANTATION
Under general anesthesia (methoxyflurane; Pittmann and Moores, Mundelein, IL), and using sterile
FIGURE 4. Photomicrograph shows an intense blue-green color, indicating collagen present in both the bone and cartilage matrix. (Masson’s trichrome; original magnification ⫻200).
surgical techniques, athymic male mice (nu/nu) (MGH, Boston, MA) 4 to 6 weeks of age each received 1 polymer construct in a dorsal subcutaneous pockets according to a random group assignment. One control group (group I) and 2 experimental groups (groups II and III) were studied. The control group (n ⫽ 6) consisted of animals receiving scaffolds saturated with calcium alginate and pluronic F127 alone, but not containing cells. Group II (n ⫽ 6) consisted of animals receiving scaffolds seeded with osteoblasts and chondrocytes in saline without the hydrogels calcium alginate and pluronic F127. Group III (n ⫽ 6) consisted of animals receiving scaffolds saturated
189
WENG ET AL
with the hydrogels alginate and pluronic F127 seeded with the 2 cell types, as previously described. SAMPLE HARVESTING
The animals were killed 12 weeks after implantation with an overdose of inhalation anesthesia. Specimens were dissected free of surrounding soft tissue and fixed in 10% phosphate-buffered formalin (Fisher Scientific, Pittsburgh, PA) for gross and histologic analysis. HISTOLOGIC ANALYSIS
After fixation in 10% phosphate-buffered formalin for 24 hours, the specimens were embedded in paraffin and sectioned. Using standard histologic techniques, serial sections were stained with hematoxylin and eosin, Safranin-O, Masson’s trichrome, and Von Kossa’s stain.
Results None of the implants showed extrusion or infection. Group I (control group) specimens showed no evidence of bone and cartilage formation. Groups II and III differed significantly in the overall 3-dimensional appearance of the explants. Specimens from group II showed only rudimentary 3-dimensional structures. These specimens were smaller and had an irregular shape when compared with specimens obtained from group III. Specimens excised from group III displayed a morphology nearly identical to that of the initial implant and mimicked the complex configuration of the original adult human TMJ condylar process (Fig 1). Specimens in both groups II and III showed evidence of new bone and cartilage formation on gross examination. Histologic examination using a standard hematoxylin and eosin (H&E) stain also showed bone and cartilage formation in these groups. In group III, a 2- to 3-mm coating of firm, white, opaque cartilage was noted on the articulating surface, which was adherent to the underlying bone. Microscopic evaluation with H&E and Safranin-O stains showed hyaline cartilage closely resembling native articular cartilage. Several sections showed the hyaline cartilage to be organized in 3 distinct layers: a superficial zone, a transitional zone, and an inferior radial zone with columnar organization of the chondrocytes. The cartilage was also organized into lobules with round, angular lacunae each containing a single chondrocyte. Abutting the cartilage was trabecular bone lined with osteoblasts (Fig 2). Microscopic evaluation using polarized light showed organized trabecular bone, with many areas showing organized concentric lamellae. The Von Kossa stain showed mineralization as the cartilage abutted
the bone. Chondrocytes could be seen infiltrating the underlying osseous substrate, forming a cartilaginousosseous interface. Ossification was located primarily in the center of the mature bone in which collagen bundles organized into bone lamella. Osteocytes in their lacunae were surrounded by bone matrix. Safranin-O stains of tissue sections from the group III specimens showed purple and green staining of bone/cartilage consistent with the presence of proteoglycan and glycosaminoglycans, such as chondroitin sulfate (Fig 3). Masson’s trichrome stain in group III showed the presence of collagen and suggested the presence of type I and II collagen, respectively, for bone and cartilage (Fig 4). Occasional remnants of polymer were seen in some areas.
Discussion This study shows that osteoblasts and chondrocytes can form in in vivo composites of bone and cartilage that may be suitable for condylar reconstruction. Only implants seeded with cells in hydrogel replicated the complex configuration of the human mandibular condyle. This suggests that the hydrogels may improve the potential of the scaffolds to maintain a specific shape. This study showed that tissues can be successfully engineered to generate composites of bone/cartilage for condyle replacement by using a combination of synthetic scaffolds into which cells are seeded at appropriate concentrations. Future studies are needed to investigate the physical properties in a controlled manner using biomechanical and dynamic force protocols. Articular chondrocytes, rather than cells from fibrocartilage, were used to avoid the rapid overgrowth of fibroblasts in tissue culture associated with the use of chondrocytes from fibrocartilage. Articular cartilage contains only chondrocytes and thus avoids fibroblast overgrowth.15 Of great interest is that the fabricated framework was of a clinically suitable size. This demonstrates that sufficient tissue can be generated when properly engineered.
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