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Use of cultivated osteoprogenitor cells to increase bone formation in segmental mandibular defects: an experimental pilot study in sheep
Int. J. Oral Maxillofac. Surg. 2001; 30: 531–537 doi:10.1054/ijom.2001.0164, available online at http://www.idealibrary.com on
Research paper: Osteobiology
Use of cultivated osteoprogenitor cells to increase bone formation in segmental mandibular defects: an experimental pilot study in sheep
H. Schliephake1, J. W. Knebel2, M. Aufderheide2, M. Tauscher3 1
Department of Oral and Maxillofacial Surgery, Georg-August University Goettingen, Goettingen, Germany 2 In vitro Toxicology, Fraunhofer Institute for Toxicology and Aerosol Research, Hannover, Germany 3 Department of Experimental Pathology, Hannover Medical School, Hannover, Germany
H. Schliephake, J. W. Knebel, M. Aufderheide, M. Tauscher: Use of cultivated osteoprogenitor cells to increase bone formation in segmental mandibular defects: an experimental pilot study in sheep. Int. J. Oral Maxillofac. Surg. 2001; 30: 531–537. 2001 International Association of Oral and Maxillofacial Surgeons Abstract. The hypothesis of the present experimental pilot study was that autogeneous cultivated osteoprogenitor cells in porous calcium phosphate scaffolds can increase bone formation in segmental defects of the mandible. The autogenous osteoprogenitor cells of eight sheep were cultivated from bone biopsies from the iliac crest and seeded into cylindrical scaffolds of pyrolized bovine bone of an overall length of 35 mm and 13 mm in diameter. Segmental defects of 35 mm length were created unilaterally in the mandibles of the animals. Reconstruction was performed using cylinders with cultivated osteoprogenitor cells in four animals and empty scaffolds in the remaining four sheep, which served as controls. After 5 months, the mandibles were retrieved and the reconstructed areas were analyzed by qualitative and quantitative histology in serial undecalcified thick-section specimens. There was significantly more bone formation in the group that had received scaffolds with cultivated bone cells (P=0.028). Bone formation was present in 34.4% of the evaluated cross-sectional units in the seeded scaffolds, while it was found in 10.4% in the control group. Although the spatial distribution of bone formation was significantly different across the scaffold in both groups, osteoprogenitor cells appeared to have increased bone formation, particularly in the centre of the defect when compared to the control group. It is concluded that the repair of segmental defects of the mandible can be enhanced by the transplantation of autogenous osteoprogenitor cells in a porous calcium phosphate scaffold.
The current trend in reconstruction of the facial skeleton is directed towards the enhancement of bone regeneration to 0901-5027/01/060531+07 $35.00/0
avoid the need for harvesting of autogenous bone grafts. One of these innovative approaches is the tissue-
Key words: tissue engineering; bone formation; mandibular reconstruction; mesenchymal stem cells. Accepted for publication 24 August 2001
engineered growth of bone, by which cultivated bone cells in scaffolds are delivered to a skeletal defect to form
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bone at the site of implantation. Experimental applications, and even more the technical and biological aspects of this new technique, have been intensely researched in recent years1,7,9,10,15,22,23. Mesenchymal stem cells (MSCs) as pluripotent cells with the ability to differentiate into several phenotypes have been identified as the major source of osteogenic cells8,20,21,28. The osteogenic phenotype of cultivated MSCs is characterized by the expression of alkaline phosphatase, collagen I, osteocalcein and by mineralization of the deposited matrix through formation of ‘bone nodules’. The technique of isolation of MSCs, the mode of cultivation and the addition of certain mitogenic factors to the culture medium can affect proliferation and the direction of differentiation into the desired phenotype21,26,32,33. This osteogenic differentiation has to be maintained during the subsequent steps of cell seeding into scaffolds and transplantation into the defect. Several factors, such as the surface and chemistry of the scaffold material and the conditions of the in-vivo environment may affect the ability of these cells to finally produce bone in vivo17,27,34. Xenogenic transplantation of committed osteoprogenitor cells into subcutaneous tissues and muscle pouches of immunocompromized rodents resulted in heterotopic bone formation and thus has proven the principal feasibility of this concept7,13,30,33. One of the major questions, however, is the practicability of tissue-engineered growth of bone in clinically relevant defects, as this setting requires high numbers of cells in large volume scaffolds. Thus, the in vitro conditions regarding culturing and seeding of osteoprogenitor cells and the in vivo environment required to maintain osteogenic differentiation and proliferation of the grafted cells are supposed to be very demanding. It was therefore the aim of the present pilot study to examine the ability of cultivated autogenous osteoprogenitor cells to enhance the repair of segmental defects of the mandible in a clinically relevant setting. Material and methods Eight adult female sheep were used for this experiment (average weight 71.5 kg). Bone samples for cultivation of osteoprogenitor cells were obtained under general anaesthesia from each animal by harvesting approximately 1 ccm of cancellous bone from the iliac crest. During
this procedure, the first premolars on both sides of the mandibles were extracted in order to allow for the creation of a large segmental defect later on that does not communicate with the oral cavity. The bone samples were placed immediately into transport medium consisting of RPMI 1640 with 100 units/ml penicillin and 100 g/ml streptomycin (Gibco). The bone pieces were then fractured into small fragments and placed into 21 cm2 dishes (Falcon) containing culture medium RPMI 1640 supplemented with fetal calf serum (10%) (Biochrom) and antibiotics (penicillin/streptomycin). The cells were then cultured under 98% humidity, 5% CO2, 37C until a subconfluent state was achieved. During this period, the medium was exchanged every second day. The cultures were then harvested by trypsinization (0.12% trypsin, 0.02% EDTA in 1:2 v/v PBS) and divided into two portions. A small portion (2104– 1105 cells) was seeded on glass trays for characterization, while the remaining cells (1106–5106 cells) were seeded on to cylinders of pyrolized bovine bone (20 mm length, 13 mm diameter, Merck, Darmstadt, Germany). This material was fabricated from bovine cancellous bone at temperatures of 1100–1300C, which had removed all organic material from the remaining calcium phosphate scaffold. The pore size ranged from 150–1000 m35. In order to maximize the degree of attachment of the cells to the scaffold, custom-made glass containers were used to accommodate the scaffold cylinders during the seeding procedure and to contain the cells in the solution as close to the scaffold surface as possible. After trypsinization, the cells were pelleted by short-time centrifugation, resuspended and administered to the cylinders by dripping the cells in the medium on to the cylinders. The scaffolds were cultivated for another 2 days to allow the cells to become attached and then were delivered to the operation room for implantation into the defects. Two cylinders were used for SEM control of the seeding procedure. The cells on the glass trays were submitted to immunohistochemical characterization of osteogenic markers. All cells showed positive staining for alkaline phosphatase, collagen I and osteocalcein (Fig. 1). Scanning electron microscopic images showed polygonal cells attached to the surface of the scaffolds. In some areas there was even a subconfluent layer of cells visible (Fig. 2).
Fig. 1. Immunohistochemical proof of collagen I expression (Immuno-Gold-Stain, original magnification 100).
Fig. 2. Scanning electron micrograph showing a subconfluent layer of osteoprogenitor cells on the scaffold surface.
The surgical procedures were carried out under general anaesthesia and endotracheal intubation. The mandibles were exposed through a submandibular incision and the edentulous portions of the mandible between the second premolar and the anterior teeth were resected unilaterally. The resulting defects of approximately 35 mm length were stabilized with bridging plates (Synthes, Waldenburg, Switzerland) and filled with cylinders of bovine bone (Fig. 3). Four animals received cylinders that had been seeded with autogenous osteoprogenitor cells and the remaining four animals served as controls, receiving empty cylinders that had only been soaked with culture medium. After wound closure, the defects were left to heal for 5 months until the mandibular segments were retrieved for histologic evaluation. Immediately after retrieval, the mandibles were fixated in 4% buffered formalin (pH 7.7) for 3 weeks. The bone segments were then trimmed, embedded into methylmethacrylate and sectioned with a diamondedged blade of a rotating saw into serial undecalcified specimens of approximately 35 m thickness (Leitz, Hamburg, Germany) perpendicular to the long axis of the cylinders. Due to the thickness of the saw blade, approximately 300 m per section were lost, so that approximately 26–32 serial crosssections per scaffold were available for
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the varying numbers of evaluated crosssections per defect were normalized to 30 sections by interpolation. Every six sections of the 30 cross-sections were averaged creating two marginal sections, two intermediate sections and one central section of the defect. The variation of bone content in these five sections was analyzed in both groups by Friedman tests. All tests were performed at a significance level of P<0.05.
Results Clinical and radiographic findings Fig. 3. Intraoperative situation. Reconstruction of the mandible by seeded bovine bone cylinders and a metal plate.
examination. Histologic specimens were surface stained with alizarine-methylene blue. Quantitative assessment was performed by dividing each cross-section of the scaffolds into 21 squares. The presence or absence of bone and cartilage was registered in each square and each square was counted as occupied if the lower and left border of the square were superimposed on bone or cartilage tissue. This avoided overcounting of only partially filled squares. Bone formation was expressed as a percentage of squares containing bone tissue or cartilage for each cross-section. Only the area of the scaffolds was evaluated; surrounding callus formation was not included. This procedure was chosen in order to rapidly acquire quantitative data of bone and cartilage formation and at the same time assess the spatial distribution of the newly formed bone across the whole scaffold. It should be stressed at this point that this method is not suitable to assess the volume density of bone tissue volume inside the scaffolds, but merely the percentage of counted squares in which bone was found. It is not meant as a substitute for point counting according to stereological principles but constitutes a scale to compare the results on an objective level. The possible error of this procedure can be assessed by relating the number of positive counts to the number of all counts by the equation36
where E is the relative error, is the proportion of the tissue under assessment and n is the total number of counts. If bone tissue in the present material was assumed to vary between
10% and 30% on average and the number of counts per animal were considered to be 600 (21 squares in approximately 30 cross-sections per animal), the relative error is supposed to vary between 0.083 and 0.042, i.e. 4.2–8.2% of the quantitative results. The percentages of all positive squares of each animal were averaged in the two groups and the mean values of the control and experimental groups were compared by Mann–Whitney test. To compare the spatial distribution of bone tissue inside the scaffolds, the values of
In the group of animals with seeded scaffolds, radiographic evaluation showed that two plates were fractured and three screws had become loose. No infection occurred in the mandibles with fractured plates. Some callus formation was seen in the defect area, which was increased in those mandibles with plate fracture. The mandibles showed a fairly well-restored contour around the blocks (Fig. 4B). In the control group, one animal presented with an infection after 6 weeks and had to be omitted from the evaluation, leaving three animals in this group. The remaining animals underwent clinically uneventfull healing. Radiographic examination showed that
Fig. 4. (A) Radiograph of a reconstructed mandible using seeded cylinders and a metal plate 5 months postoperatively. (B) Radiograph of a mandible from the control group. Note extensive callus formation and loose screws.
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higher numbers of screws had become loose and there was considerable formation of mechanically induced callus (Fig. 4A). Histologic results
The group with seeded scaffolds exhibited extensive bone formation in those areas of cylinders that faced the local mandibular bone (Fig. 5A). Most of the pores of the scaffolds were filled with newly formed bone, which showed commencing remodelling into compact lamellar bone. Bone formation decreased with increasing distance to the mandibular bone (Fig. 5B). In the central sections of the defect, the newly formed bone exhibited a looser structure than at the periphery facing the mandibular bone. Deposition of new bone had started from the surface of the scaffolds with osteoblasts and osteoid seams, indicating ongoing osteogenic activity (Fig. 5C and D). The remaining space within the pores was filled with highly vascularized soft tissue, which became more prominent towards the centre of the defect. In three of the four animals, cartilage formation was evident in the centre of the defects. This cartilage had been formed in contact with the scaffold surface and occurred both in the periphery and in the centre of the cylinders (Fig. 5D). In the control group, the areas close to the resected host bone exhibited bone ingrowth from the mandible (Fig. 6A), while the centre of the defects was filled mainly with fibrous tissue. All implanted scaffolds were separated from the surrounding callus tissue by an intervening layer of fibrous tissue (Fig. 6B). In few locations, small islands of regenerating bone were seen, with bone formation starting from the surface of the scaffolds. Bone formed in these areas was tiny and immature, and was surrounded by highly cellular soft tissue (Fig. 6C). Quantitative results
In the group with seeded scaffolds, 34.4% of the evaluated surface units exhibited bone formation on average (SD 14.7) (Fig. 7A). The distribution of newly formed bone across the defect was variable. The marginal sections of the defect exhibited bone tissue in 43.5% and 46.5% of evaluated units. These values decreased in the intermediate and central sections to 19.7%, 23.8% and 20.3% of the scaffolds, respectively (Fig. 7B). The difference in bone content of the five
Fig. 5. (A) Micrograph showing increased bone formation between the scaffolds and the local bone in the transition area. Note condensation of bone (empty arrows) between two fragments of the scaffold. Full arrows indicate the position of the reconstruction plate (AlizarineMethylene Blue, original magnification 6.3). (B) Micrograph showing bone formation in the scaffolds in central sections of the defects. Loose bone structure filled most of the crosssectional area. Full arrows indicate the position of the reconstruction plate (AlizarineMethylene Blue, original magnification 6.3). (C) Micrograph showing higher magnification of newly formed bone in the central defect sections with osteoblast seams, osteoclastic resorption and vessels in the vicinity of the newly formed bone. (Alizarine-Methylene Blue, original magnification 80). (D) Micrograph showing cartilage formation in the scaffolds in the central sections. (Alizarine-Methylene Blue, original magnification 20).
sections was significant (P=0.002). Formation of cartilage occurred in the centre of the defect at 4.8–23.8% of the evaluated units. On average there was cartilage in 1.6% (SD 1.1) of all evaluated units. The control group exhibited 10.4% of units positive for bone. The marginal sections had the highest percentage of bone fill with 19.5% and 22.4% of the evaluated units. The intermediate and central sections showed very low values with the presence of bone tissue in only 0.2%, 3.0% and 3.7% of the evaluated units (Fig. 7C). Cartilage formation was negligible in the control group. Only in one cross-section of one scaffold was there a small amount of cartilage visible; on average cartilage occupied 0.05% of the evaluated units. The difference in extent of bone formation between the group with seeded scaffolds and the control group was statistically significant (P=0.028); the distribution of the newly formed bone in the five sections of the control group was also significant (P=0.000). The extent of cartilage
formation was significantly different too (P=0.031). Discussion The results of the present study show that the extent of bone regeneration in calcium phosphate scaffolds in segmental defects of the sheep mandible can be enhanced by the presence of cultivated autogenous osteoprogenitor cells. This parallels the findings of previous reports on mesenchymal stem cells implanted into segmental defects of canine, feline and rat femura6,7,22. The effect of the expanded bone cells in the present study appears to be comparable to that reported by B et al.6 and K et al.22 In all studies, the carriers alone have provided an osteoconductive scaffold and resulted in bone formation in the defect to a certain degree, but the additional transplantation of committed osteoprogenitor cells had significantly increased the amount of bone formed inside the porous carriers. Thus, the contribution of the local host bone remains
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Fig. 6. (A) Micrograph showing bone formation in the scaffolds of the control group in the transition area to the local bone (Alizarine-Methylene Blue, original magnification 6.3). (B) Micrograph showing an envelope of fibrous tissue surrounding the scaffolds of the control group. Only small islands of bone formation can be seen (empty arrows) (Alizarine-Methylene Blue, original magnification 6.3). (C) Micrograph showing higher magnification of newly formed bone in the empty scaffolds. Little osteoblastic activity is seen. The bone is surrounded by highly cellular and vascularized soft tissue (Alizarine-Methylene Blue, original magnification 36).
Fig. 7. (A) Quantitative evaluation of bone and cartilage formation in the scaffolds (Means and standard deviation). (B) Distribution of bone in empty scaffolds. Bars represent mean values and standard deviation of the five sections, superimposed by individual values of the 30 cross sections. (C) Distribution of bone in scaffolds loaded with osteoprogenitor cells. Bars represent mean values and standard deviation of the five sections, superimposed by individual values of the 30 cross sections.
to be identified, particularly as—like in the present study—both authors have reported a collar of callus tissue that had formed around the implanted cylinders6,22. The contribution of this bone collar to bone formation inside the scaffolds is unclear, but its presence had been attributed to the osteogenic potential of the seeded osteoprogenitor cells as it was observed only in the group with loaded cylinders6,22. In the present study, a cuff of callus tissue has also been observed with the empty cylinders. This bone, however, was separated from the bone inside the cylinders by a continuous layer of fibrous tissue, indicating that the occurrence of this bone had mainly mechanical reasons due to the high frequency of screw loosening in this group and was formed independently from the regeneration inside the scaffolds. In contrast, the collar in the group of seeded scaffolds was thinner and in contact with the implanted cylinders in some areas, which may be accounted for by the fact that most of the cultivated osteoprogenitor cells were
located at the periphery of the scaffolds and have contributed to the formation of bone tissue at the surface of the cylinders. The origin of the regenerated bone may be assumed from the fate of the grafted cells. Transfection of the grafted cells with a reporter gene can visualize the surviving cells inside the newly formed bone and if the transgene has been introduced into the genome of the transplanted cells, it may also allow for estimation of proliferation after grafting23. However, definite assessment of paracrine effects on surrounding host cells is difficult to achieve, even by these highly sophisticated means. The positive effect of cultivated bone cells on bone formation inside the scaffolds was also visible from the fact that albeit significant differences between the five sections of the defects in both groups existed, spatial distribution of the newly formed bone was more homogenous across the scaffolds when compared to the control group. Beyond the observed beneficial effect of the osteo-
progenitor cells, however, questions remain as to whether the efficacy of the procedure is sufficient for clinical use and whether the degree of osteogenesis can be further enhanced, since the amount of bone formed inside the scaffolds may not be strong enough to bear functional loading of the reconstructed mandible. One of the crucial points of the technique is the number of mesenchymal stem cells that are transplanted3,4,29. Although the scanning electron micrographs of the scaffolds in the present study have shown a subconfluent state of cells on the surface of the scaffold, there are supposed to be areas inside the scaffold that are not completely covered by the up to 5106 cultured osteoblast-like cells. A cell gain of up to 108–109 osteoprogenitor cells from culture expansion and a matrix that allowed for more dense seeding would be preferrable for these purposes. High numbers of cells in large volume scaffolds, however, may be at risk. An insufficient supply with oxygen and nutritional agents, particularly in the
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centre of the seeded implant, may give rise to difficulties in maintaining the desired level of differentiation. Cartilage formation in the centre of the carriers containing osteoprogenitor cells in the present study may reflect such a change in differentiation from the osteogenic to the chondrogenic lineage. The structure and chemical composition of a cell carrier can also affect the behaviour of seeded cells and may predetermine the kind of tissue under construction. Cartilage cells are growing favourably on hyaluronan-based scaffolds and polygalactin meshes2,19,30,31; bone cells apparently grow well on ceramic carriers, collagen sponges and polygalactin fleeces12,30,33. The enhancement of proliferation and the stabilization of differentiation of the culture-expanded cells could be accomplished by the addition of external growth factors at the time of seeding. However, these growth factors will be effective only for a short period after transplantation in vivo. In order to provide a long-term supplementation with osteogenic growth factors to improve the osteogenic potential of cell/carrier constructs, the transfer of gene sequences to the transplanted cells has been used, which code for the proliferation and differentiation of bone tissue. There are a few studies reporting on the use of such cell-mediated gene therapy in skeletal reconstruction14,16,18,24,25,33. They have shown that genetically engineered mesenchymal cells did not only stimulate the transplanted cells but also the local host cells, so that a part of the resulting osteogenesis is originating from the recipient site18. Experimental studies for tissue-engineered tendon repair have used cells mainly as vehicles to deliver mitogenic and morphogenic growth factors to an area otherwise devoid of growth factors24 or to enhance the natural regeneration of local tissue in meniscus repair3. The desired function of the cells in the implanted scaffolds is different in the repair of skeletal defects, if the grafted material is expected to mimic autogenous bone grafts as the ‘gold standard’ in this field. As bone grafts both exert an intrinsic osteogenetic activity originating from its cells and at the same time recruit or activate local progenitor cells through signals incorporated in the graft matrix11, tissue-engineered grafting material in skeletal reconstructions is supposed to fulfill both tasks. At present, however, the laboratory-based cell/carrier constructs are not able to
exert such an intrinsic graft function on their own, as the cultured cells are not incorporated into a graft matrix but depend completely on the milieu and the cytokines of the recipient site to maintain their delicate state of differentiation. Therefore, the construction of tissueengineered grafts, which are biologically equivalent to natural autogenous tissue grafts, requires increased efforts in in vitro culturing and tissue construction. In conclusion, the present study has shown that the repair of segmental defects of the mandible can be enhanced by the transplantation of autogenous osteoprogenitor cells in a porous calcium phosphate scaffold. In order to obtain tissue-engineered grafts that are fully equivalent to fresh autogenous natural bone grafts, however, advances in in vitro technologies for culturing of bone cells and matrix production are required to produce grafting material that also provides matrix signals to the seeded and local cells.
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Research paper: Osteobiology
Use of cultivated osteoprogenitor cells to increase bone formation in segmental mandibular defects: an experimental pilot study in sheep
H. Schliephake1, J. W. Knebel2, M. Aufderheide2, M. Tauscher3 1
Department of Oral and Maxillofacial Surgery, Georg-August University Goettingen, Goettingen, Germany 2 In vitro Toxicology, Fraunhofer Institute for Toxicology and Aerosol Research, Hannover, Germany 3 Department of Experimental Pathology, Hannover Medical School, Hannover, Germany
H. Schliephake, J. W. Knebel, M. Aufderheide, M. Tauscher: Use of cultivated osteoprogenitor cells to increase bone formation in segmental mandibular defects: an experimental pilot study in sheep. Int. J. Oral Maxillofac. Surg. 2001; 30: 531–537. 2001 International Association of Oral and Maxillofacial Surgeons Abstract. The hypothesis of the present experimental pilot study was that autogeneous cultivated osteoprogenitor cells in porous calcium phosphate scaffolds can increase bone formation in segmental defects of the mandible. The autogenous osteoprogenitor cells of eight sheep were cultivated from bone biopsies from the iliac crest and seeded into cylindrical scaffolds of pyrolized bovine bone of an overall length of 35 mm and 13 mm in diameter. Segmental defects of 35 mm length were created unilaterally in the mandibles of the animals. Reconstruction was performed using cylinders with cultivated osteoprogenitor cells in four animals and empty scaffolds in the remaining four sheep, which served as controls. After 5 months, the mandibles were retrieved and the reconstructed areas were analyzed by qualitative and quantitative histology in serial undecalcified thick-section specimens. There was significantly more bone formation in the group that had received scaffolds with cultivated bone cells (P=0.028). Bone formation was present in 34.4% of the evaluated cross-sectional units in the seeded scaffolds, while it was found in 10.4% in the control group. Although the spatial distribution of bone formation was significantly different across the scaffold in both groups, osteoprogenitor cells appeared to have increased bone formation, particularly in the centre of the defect when compared to the control group. It is concluded that the repair of segmental defects of the mandible can be enhanced by the transplantation of autogenous osteoprogenitor cells in a porous calcium phosphate scaffold.
The current trend in reconstruction of the facial skeleton is directed towards the enhancement of bone regeneration to 0901-5027/01/060531+07 $35.00/0
avoid the need for harvesting of autogenous bone grafts. One of these innovative approaches is the tissue-
Key words: tissue engineering; bone formation; mandibular reconstruction; mesenchymal stem cells. Accepted for publication 24 August 2001
engineered growth of bone, by which cultivated bone cells in scaffolds are delivered to a skeletal defect to form
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bone at the site of implantation. Experimental applications, and even more the technical and biological aspects of this new technique, have been intensely researched in recent years1,7,9,10,15,22,23. Mesenchymal stem cells (MSCs) as pluripotent cells with the ability to differentiate into several phenotypes have been identified as the major source of osteogenic cells8,20,21,28. The osteogenic phenotype of cultivated MSCs is characterized by the expression of alkaline phosphatase, collagen I, osteocalcein and by mineralization of the deposited matrix through formation of ‘bone nodules’. The technique of isolation of MSCs, the mode of cultivation and the addition of certain mitogenic factors to the culture medium can affect proliferation and the direction of differentiation into the desired phenotype21,26,32,33. This osteogenic differentiation has to be maintained during the subsequent steps of cell seeding into scaffolds and transplantation into the defect. Several factors, such as the surface and chemistry of the scaffold material and the conditions of the in-vivo environment may affect the ability of these cells to finally produce bone in vivo17,27,34. Xenogenic transplantation of committed osteoprogenitor cells into subcutaneous tissues and muscle pouches of immunocompromized rodents resulted in heterotopic bone formation and thus has proven the principal feasibility of this concept7,13,30,33. One of the major questions, however, is the practicability of tissue-engineered growth of bone in clinically relevant defects, as this setting requires high numbers of cells in large volume scaffolds. Thus, the in vitro conditions regarding culturing and seeding of osteoprogenitor cells and the in vivo environment required to maintain osteogenic differentiation and proliferation of the grafted cells are supposed to be very demanding. It was therefore the aim of the present pilot study to examine the ability of cultivated autogenous osteoprogenitor cells to enhance the repair of segmental defects of the mandible in a clinically relevant setting. Material and methods Eight adult female sheep were used for this experiment (average weight 71.5 kg). Bone samples for cultivation of osteoprogenitor cells were obtained under general anaesthesia from each animal by harvesting approximately 1 ccm of cancellous bone from the iliac crest. During
this procedure, the first premolars on both sides of the mandibles were extracted in order to allow for the creation of a large segmental defect later on that does not communicate with the oral cavity. The bone samples were placed immediately into transport medium consisting of RPMI 1640 with 100 units/ml penicillin and 100 g/ml streptomycin (Gibco). The bone pieces were then fractured into small fragments and placed into 21 cm2 dishes (Falcon) containing culture medium RPMI 1640 supplemented with fetal calf serum (10%) (Biochrom) and antibiotics (penicillin/streptomycin). The cells were then cultured under 98% humidity, 5% CO2, 37C until a subconfluent state was achieved. During this period, the medium was exchanged every second day. The cultures were then harvested by trypsinization (0.12% trypsin, 0.02% EDTA in 1:2 v/v PBS) and divided into two portions. A small portion (2104– 1105 cells) was seeded on glass trays for characterization, while the remaining cells (1106–5106 cells) were seeded on to cylinders of pyrolized bovine bone (20 mm length, 13 mm diameter, Merck, Darmstadt, Germany). This material was fabricated from bovine cancellous bone at temperatures of 1100–1300C, which had removed all organic material from the remaining calcium phosphate scaffold. The pore size ranged from 150–1000 m35. In order to maximize the degree of attachment of the cells to the scaffold, custom-made glass containers were used to accommodate the scaffold cylinders during the seeding procedure and to contain the cells in the solution as close to the scaffold surface as possible. After trypsinization, the cells were pelleted by short-time centrifugation, resuspended and administered to the cylinders by dripping the cells in the medium on to the cylinders. The scaffolds were cultivated for another 2 days to allow the cells to become attached and then were delivered to the operation room for implantation into the defects. Two cylinders were used for SEM control of the seeding procedure. The cells on the glass trays were submitted to immunohistochemical characterization of osteogenic markers. All cells showed positive staining for alkaline phosphatase, collagen I and osteocalcein (Fig. 1). Scanning electron microscopic images showed polygonal cells attached to the surface of the scaffolds. In some areas there was even a subconfluent layer of cells visible (Fig. 2).
Fig. 1. Immunohistochemical proof of collagen I expression (Immuno-Gold-Stain, original magnification 100).
Fig. 2. Scanning electron micrograph showing a subconfluent layer of osteoprogenitor cells on the scaffold surface.
The surgical procedures were carried out under general anaesthesia and endotracheal intubation. The mandibles were exposed through a submandibular incision and the edentulous portions of the mandible between the second premolar and the anterior teeth were resected unilaterally. The resulting defects of approximately 35 mm length were stabilized with bridging plates (Synthes, Waldenburg, Switzerland) and filled with cylinders of bovine bone (Fig. 3). Four animals received cylinders that had been seeded with autogenous osteoprogenitor cells and the remaining four animals served as controls, receiving empty cylinders that had only been soaked with culture medium. After wound closure, the defects were left to heal for 5 months until the mandibular segments were retrieved for histologic evaluation. Immediately after retrieval, the mandibles were fixated in 4% buffered formalin (pH 7.7) for 3 weeks. The bone segments were then trimmed, embedded into methylmethacrylate and sectioned with a diamondedged blade of a rotating saw into serial undecalcified specimens of approximately 35 m thickness (Leitz, Hamburg, Germany) perpendicular to the long axis of the cylinders. Due to the thickness of the saw blade, approximately 300 m per section were lost, so that approximately 26–32 serial crosssections per scaffold were available for
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the varying numbers of evaluated crosssections per defect were normalized to 30 sections by interpolation. Every six sections of the 30 cross-sections were averaged creating two marginal sections, two intermediate sections and one central section of the defect. The variation of bone content in these five sections was analyzed in both groups by Friedman tests. All tests were performed at a significance level of P<0.05.
Results Clinical and radiographic findings Fig. 3. Intraoperative situation. Reconstruction of the mandible by seeded bovine bone cylinders and a metal plate.
examination. Histologic specimens were surface stained with alizarine-methylene blue. Quantitative assessment was performed by dividing each cross-section of the scaffolds into 21 squares. The presence or absence of bone and cartilage was registered in each square and each square was counted as occupied if the lower and left border of the square were superimposed on bone or cartilage tissue. This avoided overcounting of only partially filled squares. Bone formation was expressed as a percentage of squares containing bone tissue or cartilage for each cross-section. Only the area of the scaffolds was evaluated; surrounding callus formation was not included. This procedure was chosen in order to rapidly acquire quantitative data of bone and cartilage formation and at the same time assess the spatial distribution of the newly formed bone across the whole scaffold. It should be stressed at this point that this method is not suitable to assess the volume density of bone tissue volume inside the scaffolds, but merely the percentage of counted squares in which bone was found. It is not meant as a substitute for point counting according to stereological principles but constitutes a scale to compare the results on an objective level. The possible error of this procedure can be assessed by relating the number of positive counts to the number of all counts by the equation36
where E is the relative error, is the proportion of the tissue under assessment and n is the total number of counts. If bone tissue in the present material was assumed to vary between
10% and 30% on average and the number of counts per animal were considered to be 600 (21 squares in approximately 30 cross-sections per animal), the relative error is supposed to vary between 0.083 and 0.042, i.e. 4.2–8.2% of the quantitative results. The percentages of all positive squares of each animal were averaged in the two groups and the mean values of the control and experimental groups were compared by Mann–Whitney test. To compare the spatial distribution of bone tissue inside the scaffolds, the values of
In the group of animals with seeded scaffolds, radiographic evaluation showed that two plates were fractured and three screws had become loose. No infection occurred in the mandibles with fractured plates. Some callus formation was seen in the defect area, which was increased in those mandibles with plate fracture. The mandibles showed a fairly well-restored contour around the blocks (Fig. 4B). In the control group, one animal presented with an infection after 6 weeks and had to be omitted from the evaluation, leaving three animals in this group. The remaining animals underwent clinically uneventfull healing. Radiographic examination showed that
Fig. 4. (A) Radiograph of a reconstructed mandible using seeded cylinders and a metal plate 5 months postoperatively. (B) Radiograph of a mandible from the control group. Note extensive callus formation and loose screws.
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higher numbers of screws had become loose and there was considerable formation of mechanically induced callus (Fig. 4A). Histologic results
The group with seeded scaffolds exhibited extensive bone formation in those areas of cylinders that faced the local mandibular bone (Fig. 5A). Most of the pores of the scaffolds were filled with newly formed bone, which showed commencing remodelling into compact lamellar bone. Bone formation decreased with increasing distance to the mandibular bone (Fig. 5B). In the central sections of the defect, the newly formed bone exhibited a looser structure than at the periphery facing the mandibular bone. Deposition of new bone had started from the surface of the scaffolds with osteoblasts and osteoid seams, indicating ongoing osteogenic activity (Fig. 5C and D). The remaining space within the pores was filled with highly vascularized soft tissue, which became more prominent towards the centre of the defect. In three of the four animals, cartilage formation was evident in the centre of the defects. This cartilage had been formed in contact with the scaffold surface and occurred both in the periphery and in the centre of the cylinders (Fig. 5D). In the control group, the areas close to the resected host bone exhibited bone ingrowth from the mandible (Fig. 6A), while the centre of the defects was filled mainly with fibrous tissue. All implanted scaffolds were separated from the surrounding callus tissue by an intervening layer of fibrous tissue (Fig. 6B). In few locations, small islands of regenerating bone were seen, with bone formation starting from the surface of the scaffolds. Bone formed in these areas was tiny and immature, and was surrounded by highly cellular soft tissue (Fig. 6C). Quantitative results
In the group with seeded scaffolds, 34.4% of the evaluated surface units exhibited bone formation on average (SD 14.7) (Fig. 7A). The distribution of newly formed bone across the defect was variable. The marginal sections of the defect exhibited bone tissue in 43.5% and 46.5% of evaluated units. These values decreased in the intermediate and central sections to 19.7%, 23.8% and 20.3% of the scaffolds, respectively (Fig. 7B). The difference in bone content of the five
Fig. 5. (A) Micrograph showing increased bone formation between the scaffolds and the local bone in the transition area. Note condensation of bone (empty arrows) between two fragments of the scaffold. Full arrows indicate the position of the reconstruction plate (AlizarineMethylene Blue, original magnification 6.3). (B) Micrograph showing bone formation in the scaffolds in central sections of the defects. Loose bone structure filled most of the crosssectional area. Full arrows indicate the position of the reconstruction plate (AlizarineMethylene Blue, original magnification 6.3). (C) Micrograph showing higher magnification of newly formed bone in the central defect sections with osteoblast seams, osteoclastic resorption and vessels in the vicinity of the newly formed bone. (Alizarine-Methylene Blue, original magnification 80). (D) Micrograph showing cartilage formation in the scaffolds in the central sections. (Alizarine-Methylene Blue, original magnification 20).
sections was significant (P=0.002). Formation of cartilage occurred in the centre of the defect at 4.8–23.8% of the evaluated units. On average there was cartilage in 1.6% (SD 1.1) of all evaluated units. The control group exhibited 10.4% of units positive for bone. The marginal sections had the highest percentage of bone fill with 19.5% and 22.4% of the evaluated units. The intermediate and central sections showed very low values with the presence of bone tissue in only 0.2%, 3.0% and 3.7% of the evaluated units (Fig. 7C). Cartilage formation was negligible in the control group. Only in one cross-section of one scaffold was there a small amount of cartilage visible; on average cartilage occupied 0.05% of the evaluated units. The difference in extent of bone formation between the group with seeded scaffolds and the control group was statistically significant (P=0.028); the distribution of the newly formed bone in the five sections of the control group was also significant (P=0.000). The extent of cartilage
formation was significantly different too (P=0.031). Discussion The results of the present study show that the extent of bone regeneration in calcium phosphate scaffolds in segmental defects of the sheep mandible can be enhanced by the presence of cultivated autogenous osteoprogenitor cells. This parallels the findings of previous reports on mesenchymal stem cells implanted into segmental defects of canine, feline and rat femura6,7,22. The effect of the expanded bone cells in the present study appears to be comparable to that reported by B et al.6 and K et al.22 In all studies, the carriers alone have provided an osteoconductive scaffold and resulted in bone formation in the defect to a certain degree, but the additional transplantation of committed osteoprogenitor cells had significantly increased the amount of bone formed inside the porous carriers. Thus, the contribution of the local host bone remains
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Fig. 6. (A) Micrograph showing bone formation in the scaffolds of the control group in the transition area to the local bone (Alizarine-Methylene Blue, original magnification 6.3). (B) Micrograph showing an envelope of fibrous tissue surrounding the scaffolds of the control group. Only small islands of bone formation can be seen (empty arrows) (Alizarine-Methylene Blue, original magnification 6.3). (C) Micrograph showing higher magnification of newly formed bone in the empty scaffolds. Little osteoblastic activity is seen. The bone is surrounded by highly cellular and vascularized soft tissue (Alizarine-Methylene Blue, original magnification 36).
Fig. 7. (A) Quantitative evaluation of bone and cartilage formation in the scaffolds (Means and standard deviation). (B) Distribution of bone in empty scaffolds. Bars represent mean values and standard deviation of the five sections, superimposed by individual values of the 30 cross sections. (C) Distribution of bone in scaffolds loaded with osteoprogenitor cells. Bars represent mean values and standard deviation of the five sections, superimposed by individual values of the 30 cross sections.
to be identified, particularly as—like in the present study—both authors have reported a collar of callus tissue that had formed around the implanted cylinders6,22. The contribution of this bone collar to bone formation inside the scaffolds is unclear, but its presence had been attributed to the osteogenic potential of the seeded osteoprogenitor cells as it was observed only in the group with loaded cylinders6,22. In the present study, a cuff of callus tissue has also been observed with the empty cylinders. This bone, however, was separated from the bone inside the cylinders by a continuous layer of fibrous tissue, indicating that the occurrence of this bone had mainly mechanical reasons due to the high frequency of screw loosening in this group and was formed independently from the regeneration inside the scaffolds. In contrast, the collar in the group of seeded scaffolds was thinner and in contact with the implanted cylinders in some areas, which may be accounted for by the fact that most of the cultivated osteoprogenitor cells were
located at the periphery of the scaffolds and have contributed to the formation of bone tissue at the surface of the cylinders. The origin of the regenerated bone may be assumed from the fate of the grafted cells. Transfection of the grafted cells with a reporter gene can visualize the surviving cells inside the newly formed bone and if the transgene has been introduced into the genome of the transplanted cells, it may also allow for estimation of proliferation after grafting23. However, definite assessment of paracrine effects on surrounding host cells is difficult to achieve, even by these highly sophisticated means. The positive effect of cultivated bone cells on bone formation inside the scaffolds was also visible from the fact that albeit significant differences between the five sections of the defects in both groups existed, spatial distribution of the newly formed bone was more homogenous across the scaffolds when compared to the control group. Beyond the observed beneficial effect of the osteo-
progenitor cells, however, questions remain as to whether the efficacy of the procedure is sufficient for clinical use and whether the degree of osteogenesis can be further enhanced, since the amount of bone formed inside the scaffolds may not be strong enough to bear functional loading of the reconstructed mandible. One of the crucial points of the technique is the number of mesenchymal stem cells that are transplanted3,4,29. Although the scanning electron micrographs of the scaffolds in the present study have shown a subconfluent state of cells on the surface of the scaffold, there are supposed to be areas inside the scaffold that are not completely covered by the up to 5106 cultured osteoblast-like cells. A cell gain of up to 108–109 osteoprogenitor cells from culture expansion and a matrix that allowed for more dense seeding would be preferrable for these purposes. High numbers of cells in large volume scaffolds, however, may be at risk. An insufficient supply with oxygen and nutritional agents, particularly in the
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centre of the seeded implant, may give rise to difficulties in maintaining the desired level of differentiation. Cartilage formation in the centre of the carriers containing osteoprogenitor cells in the present study may reflect such a change in differentiation from the osteogenic to the chondrogenic lineage. The structure and chemical composition of a cell carrier can also affect the behaviour of seeded cells and may predetermine the kind of tissue under construction. Cartilage cells are growing favourably on hyaluronan-based scaffolds and polygalactin meshes2,19,30,31; bone cells apparently grow well on ceramic carriers, collagen sponges and polygalactin fleeces12,30,33. The enhancement of proliferation and the stabilization of differentiation of the culture-expanded cells could be accomplished by the addition of external growth factors at the time of seeding. However, these growth factors will be effective only for a short period after transplantation in vivo. In order to provide a long-term supplementation with osteogenic growth factors to improve the osteogenic potential of cell/carrier constructs, the transfer of gene sequences to the transplanted cells has been used, which code for the proliferation and differentiation of bone tissue. There are a few studies reporting on the use of such cell-mediated gene therapy in skeletal reconstruction14,16,18,24,25,33. They have shown that genetically engineered mesenchymal cells did not only stimulate the transplanted cells but also the local host cells, so that a part of the resulting osteogenesis is originating from the recipient site18. Experimental studies for tissue-engineered tendon repair have used cells mainly as vehicles to deliver mitogenic and morphogenic growth factors to an area otherwise devoid of growth factors24 or to enhance the natural regeneration of local tissue in meniscus repair3. The desired function of the cells in the implanted scaffolds is different in the repair of skeletal defects, if the grafted material is expected to mimic autogenous bone grafts as the ‘gold standard’ in this field. As bone grafts both exert an intrinsic osteogenetic activity originating from its cells and at the same time recruit or activate local progenitor cells through signals incorporated in the graft matrix11, tissue-engineered grafting material in skeletal reconstructions is supposed to fulfill both tasks. At present, however, the laboratory-based cell/carrier constructs are not able to
exert such an intrinsic graft function on their own, as the cultured cells are not incorporated into a graft matrix but depend completely on the milieu and the cytokines of the recipient site to maintain their delicate state of differentiation. Therefore, the construction of tissueengineered grafts, which are biologically equivalent to natural autogenous tissue grafts, requires increased efforts in in vitro culturing and tissue construction. In conclusion, the present study has shown that the repair of segmental defects of the mandible can be enhanced by the transplantation of autogenous osteoprogenitor cells in a porous calcium phosphate scaffold. In order to obtain tissue-engineered grafts that are fully equivalent to fresh autogenous natural bone grafts, however, advances in in vitro technologies for culturing of bone cells and matrix production are required to produce grafting material that also provides matrix signals to the seeded and local cells.
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33. S H, B H, L W, R M, M H, P H. In-vitro Manipulation und Transfektion von prima¨ ren humanen Osteoprogenitorzellen mit BMP2 fu¨ r die gewebetechnologische Herstellung von Knochen. Mund-KieferGesichtschirurgie 2000: 4: S470–S473. 34. S WJ. Bone and the hematopoetic and immune systems: a report of the proceedings of a scientific workshop. J Bone Miner Res 1998: 13: 537–543. 35. T¨ S. Die Hydroxylapatitkeramik Endobon—Eine alternative Therapiemo¨ glichkeit fu¨ r Knochendefekte. In: Venbrocks R, Salis-Goglio G, eds: Jahrbuch der Orthopa¨ die. Zu¨ rich: Biermann 1989: 231–250. 36. W ER. Morphometry of the human lung. Berlin: Springer 1963: 21. Address: Professor Henning Schliephake Direktor der Abteilung fu¨ r Mund-, Kiefer- und Gesichtschirurgie Georg-August Universita¨ t Goettingen Robert-Koch Str. 40 37075 Goettingen Germany Tel: +49 551 398306 Fax: +49 551 3912653 E-mail: [email protected]