Baixe Bone formation by three-dimensional stromal osteoblast e outras Notas de estudo em PDF para Engenharia de Produção, somente na Docsity! Bone formation by three-dimensional stromal osteoblast culture in biodegradable polymer scaffolds Susan L. Ishaug,1,† Genevieve M. Crane,1 Michael J. Miller,2 Alan W. Yasko,3 Michael J. Yaszemski,4,‡ and Antonios G. Mikos1,* 1Cox Laboratory for Biomedical Engineering, Institute of Biosciences and Bioengineering, Rice University, P.O. Box 1892, Houston, Texas 77251; 2Department of Reconstructive and Plastic Surgery, University of Texas MD Anderson Cancer Center, Houston, Texas 77030; 3Department of Orthopaedic Surgery, University of Texas MD Anderson Cancer Center, Houston, Texas 77030; 4Department of Orthopaedic Surgery, Wilford Hall and Medical Center, Lackland AFB, Texas 78236 Bone formation was investigated in vitro by culturing stro- mal osteoblasts in three-dimensional (3-D), biodegradable poly(DL-lactic-co-glycolic acid) foams. Three polymer foam pore sizes, ranging from 150–300, 300–500, and 500–710 mm, and two different cell seeding densities, 6.83 × 105 cells/cm2 and 22.1 × 105 cells/cm2, were examined over a 56-day cul- ture period. The polymer foams supported the proliferation of seeded osteoblasts as well as their differentiated function, as demonstrated by high alkaline phosphatase activity and deposition of a mineralized matrix by the cells. Cell number, alkaline phosphatase activity, and mineral deposition in- creased significantly over time for all the polymer foams. Osteoblast foam constructs created by seeding 6.83 × 105 cells/cm2 on foams with 300–500 mm pores resulted in a cell density of 4.63 × 105 cells/cm2 after 1 day in culture; they had alkaline phosphatase activities of 4.28 × 10−7 and 2.91 × 10−6 mmol/cell/min on Days 7 and 28, respectively; and they had a cell density that increased to 18.7 × 105 cells/cm2 by Day 56. For the same constructs, the mineralized matrix reached a maximum penetration depth of 240 mm from the top surface of the foam and a value of 0.083 mm for miner- alized tissue volume per unit of cross sectional area. Seeding density was an important parameter for the constructs, but pore size over the range tested did not affect cell prolifera- tion or function. This study suggests the feasibility of using poly(a-hydroxy ester) foams as scaffolding materials for the transplantation of autogenous osteoblasts to regenerate bone tissue. © 1997 John Wiley & Sons, Inc. INTRODUCTION Skeletal reconstruction is required in cases involv- ing large defects created by tumor resection, trauma, and skeletal abnormalities.1 Presently, grafts and flaps of autogenous tissue are two of the most successful means of reconstruction because they allow for the transplantation of bone containing bioactive mol- ecules, live cells, and, frequently, a vascular supply that will allow the transplant to survive and remodel even in hostile radiated environments.2 Other current therapies involve the use of allograft bone, nonde- gradable bone cement, metals, and ceramics.3 All of these options have their associated problems and limi- tations: only a minimal amount of tissue can be har- vested for autografts, and it can be very difficult to form into the desired shapes; allografts have the po- tential of transferring pathogens; and synthetic im- plants may result in stress shielding to the surround- ing bone or fatigue failure of the implant. These short- comings have inspired a search for improved methods of repairing skeletal defects. The ideal bone substitute would approximate the autograft, requiring minimally that it be biocompat- ible and osteoconductive, contain osteoinductive fac- tors to enhance new bone ingrowth, and contain os- teogenic cells to begin secreting new extracellular ma- trix.1 Bone regeneration by autogenous osteoblast transplantation meets these requirements and thus holds promise as an improved method of skeletal re- construction. The scaffolding material used in this ap- proach must allow for the attachment of osteoblasts because they are anchorage-dependent cells that re- quire a supportive matrix in order to survive.4 The *To whom correspondence should be addressed. †Present address: Los Alamos National Laboratory, MS M888, CST-1, Los Alamos, New Mexico 87545. ‡Present address: Department of Orthopedic Surgery, Mayo Clinic, Rochester, Minnesota 55905. Journal of Biomedical Materials Research, Vol. 36, 17–28 (1997) © 1997 John Wiley & Sons, Inc. CCC 0021-9304/97/010017-12 material also must provide an appropriate environ- ment for the proliferation and function of osteoblasts and allow for the ingrowth of vascular tissue to ensure the survival of the transplanted cells. Finally, it should be biodegradable with a controllable rate of degrada- tion into molecules that easily can be metabolized or excreted, and it should be processable into irregular 3-D shapes. Poly(a-hydroxy esters), including poly(DL-lactic- co-glycolic acid) (PLGA) copolymers, satisfy many, if not all, of these material requirements.5 The ability of two-dimensional (2-D) PLGA films to support osteo- blast attachment, growth, and function already has been established in our laboratory.6 Recently, we also demonstrated that osteoblasts on polymer films mi- grate from isolated osteoblast cultures and bone chips as a monolayer of cells and continue to proliferate to form multilayers.7 Two-dimensional cultures such as these were necessary to establish the potential for us- ing poly(a-hydroxy esters) as a supportive material for osteoblast growth and function, but they are not the ideal form for transplanting into defect sites. Skel- etal defects vary in size and shape and require a 3-D graft material to fill or replace the missing tissue. Poly(a-hydroxy esters) can be fabricated into 3-D foams that can serve as a supportive scaffold for the culture and transplantation of osteoblasts,8 and they have the potential of filling in skeletal defects of vari- ous sizes and shapes. These poly(a-hydroxy esters) foams already have been shown to allow for the pen- etration of vascular tissue,9 which is essential to sup- porting the metabolic needs of the transplanted cells. Osteoblasts should populate the constructs by prolif- eration of the transplanted cells and the migration of cells into the construct from the surrounding tissue while the polymer scaffold gradually degrades. Even- tually the construct will be filled with calcified extra- cellular matrix secreted by the osteoblasts and will be devoid of the synthetic biodegradable polymer. The transplanted osteoblasts may be obtained by a variety of methods for in vitro and in vivo studies, including migration from bone chips and enzymatic digestion of harvested bone.10 However, the most de- sirable method would be to obtain osteoblasts percu- taneously from the patient’s bone marrow. This would avoid the need for open surgery, with its possible do- nor-site complications of pain, infection, and damage to nerves and blood vessels. In addition, because the cells would be of autogenous origin, there would be no risk of immune rejection and little change of patho- gen transfer. Several studies have demonstrated the feasibility of obtaining bone-forming cells from hu- man, rabbit, and rat bone marrow.11–13 These tech- niques involve in vitro expansion of the mesenchymal stem cells present in the marrow by the addition to culture media of the appropriate factors to enhance osteoblast differentiation and function. An investigation of the effects of polymer foam morphology and culture conditions on cell prolifera- tion and function was needed to elucidate the impor- tant parameters in the design of an in vitro osteoblast foam culture system before osteoblast transplantation could be attempted in vivo. Such an investigation is the focus of the present study. Rat stromal osteoblasts have been seeded onto highly porous PLGA foams of different pore sizes and cell seeding densities and cul- tured over a 56-day period. This study addresses: 1) whether polymer foam pore size in the range of 150– 710 mm affects osteoblast proliferation and function in vitro, 2) whether osteoblast seeding density on poly- mer foams affects cell attachment, proliferation, and function in vitro, and 3) whether a 3-D osseous tissue can be formed by culturing osteoblasts in polymer foams in vitro. MATERIALS AND METHODS Polymer foam fabrication Polymer foams of three different pore sizes were fabricated by a solvent-casting particulate-leaching technique with NaCl as the leachable component.14 NaCl was sieved into particles ranging in diameter from 150–300, 300–500, or 500–710 mm and combined with 75:25 PLGA (Birmingham Polymers, Birming- ham, AL) dissolved in chloroform to make 90% po- rous foams of various pore sizes. The foams were ap- proximately 1.9 mm thick and cut into 7 mm diameter disks. They were stored under vacuum until use. Prior to cell seeding, the foams were prewetted with ethanol for 30 min to sterilize and enhance their water up- take.15 The ethanol was removed by soaking with agi- tation for 1 h in three changes of phosphate-buffered saline (PBS) and then for 3 h in two changes of media. Stromal cell isolation, seeding, and culture Stromal osteoblastic cells were obtained from the marrow of young adult male (6-weeks old, 150–170 g) Sprague Dawley rats.13 Following euthanasia by ethyl ether inhalation, femora were aseptically excised, cleaned of soft tissue, and washed in Dulbecco’s Modified Eagle medium (DMEM) (Life Technologies, Grand Island, NY) containing 250 mg/mL gentamicin sulfate (GS) (Sigma Chemical, St. Louis, MO). This concentration of antibiotics is 10 times the normal amount used in cell culture and was used as a precau- tionary measure to avoid contamination during the femora harvest. The metaphyseal ends then were cut off and the marrow flushed from the midshaft with 5 mL of primary media [DMEM containing 10% fetal bovine serum (FBS, Hyclone, Logan, Utah) and 25 mg/ mL GS] using a syringe equipped with a 22-gauge 18 ISHAUG ET AL. vent-casting particulate-leaching technique using salt particles as the leachable porogen. The resulting foam pore sizes were dictated by the size of the salt particles used in the fabrication process. Foams processed with salt particles sieved into the size ranges 150–300 mm [Fig. 1(a)], 300–500 mm [Fig. 1(b)], and 500–710 mm [Fig. 1(c)] exhibited pore sizes comparable to the size of the salt particles used in the fabrication process. An interconnected pore morphology was apparent in all foams, which was possible because of the high poros- ity of the scaffolds. Cell proliferation Osteoblasts seeded onto 75:25 PLGA foams attached to the pore surfaces and continued to proliferate over the 56-day in vitro culture period on all the samples producing 3-D osteoblast foam constructs. Scanning electron micrographs reveal the pore morphology of a foam created using salt particles 300–500 mm in diam- eter before [Fig. 1(b)] and after [Fig. 1(d)] osteoblast seeding. Osteoblasts can be seen covering the pore surfaces after 1 day in culture [Fig. 1(d)]. Only a fraction of the seeded cells remained at- tached to the polymer foams. The initial high-seeding density of 22.1 × 105 cells/cm2 resulted in only 11.8 × 105 cells/cm2 remaining attached to the 300–500 mm foams after one day in culture, giving a percent attach- ment of 53 ± 1% after this 24-h period. For the lower seeding density, 4.63 × 105 cell/cm2 of the 6.83 × 105 cells/cm2 seeded onto the 300–500 mm foams re- mained attached after 1 day in culture, giving a higher (p < 0.01) percent attachment of 68 ± 5%. Confocal depth projection micrographs demon- strated the initial rapid growth achieved when osteo- blasts were seeded at a low density on polymer foams having pore sizes 300–500 mm in diameter [Fig. 2(a– c)]. Proliferation results, as determined by quantifica- tion of DNA in the polymer foams, also indicated that osteoblasts grew more rapidly in the foams seeded with a lower cell density (p < 0.01 at day 1), eventually reaching comparable cell numbers to the foams seeded with a high cell density by Day 7 [Fig. 3(a)]. Comparable cell numbers also were found in the foams after 14 days in culture; however, by 28 and 56 days in culture, the number of osteoblasts found in the foams seeded with a lower cell density was lower (p < 0.05) than that for the high-density seeded foams after the same culture time. Osteoblast proliferation leveled off in all the osteoblast foam constructs studied fol- lowing 28 days in culture, with no significant change in cell numbers between Day 28 and Day 56 [Fig. 3(a,b)]. Osteoblasts proliferated equally well on poly- mer foams of all pore sizes studied [Fig. 3(b)]. The rate of osteoblast proliferation on TCPS should not be com- pared to the osteoblast foam construct rates of prolif- eration because they were seeded at a much lower cell density. ALPase activity for these cultures will be compared to activity results of the osteoblast foam constructs in the next section. Alkaline phosphatase activity All osteoblast foam cultures expressed high alkaline phosphatase activity that increased substantially over time in culture [Fig. 4(a,b)]. Osteoblasts seeded at a low-cell density on polymer foams of 300–500 mm ap- peared to express higher levels of ALPase activity compared to foams seeded with a higher cell density at all days in culture; nevertheless, these results were not significantly different [Fig. 4(a)]. Measurement of ALPase activity of osteoblasts cultured on standard TCPS were included for comparison to the 3-D sub- strates. The cells in the constructs expressed levels of comparable ALPase activity to the standard 2-D os- teoblast cultures after 7 and 14 days in culture [Fig. 4(a)], with the exception of Day 28, where slightly higher activity (p < 0.05) was observed for osteoblasts cultured on TCPS than in osteoblast foam constructs initially seeded with a high-cell density. Polymer foam pore size did not affect the expression of ALPase ac- tivity of the osteoblasts at any culture period [Fig. 4(b)]. Histology In addition to growth, the osteoblasts began to lay down osteoid, demonstrated by the pink regions of H and E-stained sections of a construct after 56 days in culture [Fig. 5(a,c)]. Osteoblasts appeared to be em- bedded in the newly formed tissue matrix, which is characteristic of the natural osteoblast differentiation and their progression into osteocytic cells. Von Kos- sa’s staining of parallel histological sections revealed that portions of the tissue matrix had been mineral- ized, with the mineral deposits predominantly cover- ing the surface of the construct [Fig. 5(b,d)]. Mineralization Vertical cross-sections of the osteoblast foam con- structs stained by von Kossa’s method, such as the one shown in Figure 5(d), were used to quantify the min- eralized tissue by histomorphometric techniques. The penetration depth of osseous tissue into the construct (distance from polymer surface to bottom of mineral- ized tissue front) was found to increase over the study 21BONE FORMATION IN POLYMER SCAFFOLDS period for all samples [Fig. 6(a)]. A maximum penetra- tion depth of mineralized tissue of 240 ± 82 mm was reached for osteoblast foam constructs of pore size 300–500 mm initially seeded with a low-cell density. The penetration depth of mineralized tissue was sig- nificantly affected (p < 0.05) by initial osteoblast seed- ing density only at day 14 in culture and was not affected by foam pore size at any time point [Fig. 6(a)]. The ratio of the mineralized tissue volume to top surface area of the foams increased dramatically for all the osteoblast foam constructs over the 56-day culture period [Fig. 6(b)]. Neither polymer foam pore size nor initial cell seeding density had any significant effect on the mineralized volume to surface area, with the ex- ception that after 14 days in culture, foams with pore sizes of 500–700 mm had a greater (p < 0.05) mineral- ized volume to surface area than foams with pore sizes of 150–300 mm. Polymer degradation All the foams degraded throughout the culture pe- riod of the study, as measured by their decrease in weight average molecular weight (Fig. 7). A similar decrease also was observed for the number average molecular weight (data not shown). Neither foam pore size (Fig. 7) nor seeding density (data not shown) sig- nificantly affected the degradation of the constructs. The average decrease in weight average molecular weight of the polymer in all the osteoblast foam con- structs from Day 1 to Day 56 was 69 ± 5%. DISCUSSION This study set out to answer the following ques- tions: 1) Do differences in polymer foam pore size in Figure 1. Scanning electron micrographs of the top surface of 90% porous 75:25 PLGA foams fabricated by a solvent-casting particulate-leaching technique using sodium chloride as the leachable component. An interconnecting porous structure was achieved in all foams. The foams were processed with the salt particles sieved into the size ranges 150–300 mm (a), 300–500 mm (b), and 500–710 mm (c) and exhibited pore sizes comparable to the salt particle size. A top view of a polymer foam prepared with salt particles ranging from 300–500 mm and seeded with high cell density (22.1 × 105 cells/cm2) reveals layers of osteoblasts covering the pores surfaces after 1 day in culture (d). 22 ISHAUG ET AL. the range of 150–710 mm affect osteoblast proliferation and function in vitro? 2) Does osteoblast seeding den- sity on polymer foams affect cell attachment, prolif- eration, and function in vitro? 3) Can a 3-D osseous tissue be formed by culturing osteoblasts in polymer foams in vitro? To answer the first question, we cultured stromal osteoblasts in polymer foams having pores in the size ranges of 150–300, 300–500, and 500–710 mm. Based on DNA content, ALPase activity, and mineral deposition in the osteoblast foam constructs, we found that dif- ferences in polymer foam pore size within a range of 150–710 mm did not significantly affect osteoblast pro- liferation or function in vitro. Pore size was investi- gated because in studies using ceramic materials19 for bone growth, an optimum pore size of 200–400 mm had been observed in vivo. Since bone is a vascular tissue, this pore-size range may be optimal because it provides sufficient space for growth of vascular tissue. One reason we may not have seen any effects based on pore size is the lack of angiogenesis (new vessel for- mation) in our in vitro culture system. It also has been suggested that the pore-size range of 200–400 mm is preferred by osteoblasts because it provides the opti- mum compression and tension on the osteoblast’s mechanoreceptors.20 Since our static culture system Figure 2. Confocal micrographs depicting the rapid initial growth rate of osteoblasts seeded with a lower cell density (6.83 × 105 cells/cm2) in 300–500 mm foams. Pictures were taken after 1 (a), 4 (c), and 7 (d) days in culture. A 300–500 mm polymer foam seeded with a higher cell density (22.1 × 105 cells/cm2) after 1 day in culture (b) reveals more cells covering the pores of the polymer than in (a). Color corresponds to the depth from the polymer surface, with red being closest to the surface and blue being at 548 mm (a), 482 mm (b), 656 mm (c), and 463 mm (d) from the surface. The bar corresponds to 250 mm in all four micrographs. 23BONE FORMATION IN POLYMER SCAFFOLDS ability to culture osteoblasts from human bone mar- row already has been established.11,16,25 Also, poly(a- hydroxy esters) already have been FDA approved for certain clinical uses, such as degradable sutures. The poly(a-hydroxy ester) used in foam construction can be tailored to degrade over periods ranging from weeks to years, depending on the clinical need.26 Re- placement of the resorbed polymer scaffold by the host bone subsequently would result in natural bone regeneration in the defect site. Although 3-D osteoblast cell culture has been dem- onstrated on a variety of matrices, such as poly(gly- colic acid) meshes,23 collagen matrices,21,27 ceramics,28 and polyphosphazenes,29 we believe the PLGA con- structs offer distinct advantages over other methods. Poly(glycolic acid) meshes inherently have low me- chanical strength, and their relatively thin 100 mm sheets make repairing larger defects more challenging; collagen matrices also are relatively weak, and their enzymatically dependent degradation could result in unpredictable degradation rates; the success of trans- planted collagen/osteoblast matrices may be compro- mised due to immunological responses to collagen obtained from xenogeneic or allogeneic sources or contaminants from transfected cell-line collagen puri- fications; the slow degradation of ceramic matrices may pose a problem for the replacement of these de- vices with new host bone and may alter the mechani- cal properties of the newly formed bone; and finally, the biocompatibility of polyphosphazenes still needs to be tested as they are fairly new materials. In con- trast, poly(lactic-co-glycolic acid) foams can be fabri- cated in any size, they degrade by hydrolysis in a controllable fashion, and they are biocompatible. In addition to in vivo regenerative potential, these Figure 6. Average maximum depth below the polymer surface that mineralized tissue was deposited (a) and total mineralized volume/surface area (b) in the osteoblast foam constructs over culture time. These values were determined by histomorphometry using vertical tissue cross sections of the constructs stained with von Kossa’s silver nitrate method [similar to that shown in Fig. 5(d)]. The mineralized volume/surface area in 3-D was taken to be proportional to the mineralized tissue area per length in the 2-D sections. Mineralization initially was detected in the foams by day 14 in culture and increased significantly throughout the 56-day culture time. No significant differences were seen between foams of different pore sizes and cell seeding densities, where HSD and LSD stand for high seeding density (22.1 × 105 cells/cm2) and low seeding density (6.83 × 105 cells/ cm2), respectively. Figure 7. Weight average molecular weights of the 75:25 PLGA polymer foams, initially seeded with a high cell den- sity (22.1 × 105 cells/cm2), over time. No significant differ- ence in the weight average molecular weight was seen among the polymer foams of different pore size. Similar results were found for the foams seeded with a lower cell density (data not shown). 26 ISHAUG ET AL. 3-D osteoblast cultures may provide a better in vitro model of osteoblast function than do conventional 2-D systems because osteoblasts are found in a three- dimensional network in vivo. More accurate responses of osteoblasts to pharmacological or mechanical stimuli thus may be determined. We presently are us- ing this culture system to investigate osteoblast re- sponses to mechanical loads in a three-dimensional environment.30 Three-dimensional osteoblast cultures also have the potential of yielding greater cellularity than do 2-D cultures. In a previous 2-D study carried out in our laboratory,6 calvarial osteoblast prolifera- tion plateaued at approximately 1 × 105 cells/cm2 on flat 75:25 PLGA films compared to cellularities rang- ing from 17–19 × 105 cells/cm2 achieved in the 3-D foams after 14 days in culture. This may be attributed to the larger surface area provided by the intercon- nected pores and the three-dimensional nature of the constructs. Similar trends between 2-D and 3-D cul- tures were found when osteoblast-like cells were cul- tured on polyphosphazene matrices.29 Direct comparisons between the present study and previous studies can be made but are difficult because of differences in initial seeding density. Three- dimensional polyphosphazene matrices (average pore size of 165 mm) supported the growth of 8 × 105 cells/ cm2 after 14 days in culture, but we produced 3-D cultures in 75:25 PLGA foams of 150–300 mm pores having 15 × 105 cells/cm2. The greater number of cells found in our 3-D constructs most likely is due to the greater 22 × 105 cells/cm2 seeding density compared to the 1 × 105 cells/cm2 density used to seed the poly- phosphazene matrices. Osteoblasts cultured on po- rous calcium phosphate ceramic28 originally seeded with approximately 2.6 × 105 cells/cm2 had 12 × 105 cells/cm2 after 70 days in culture whereas we achieved comparable osteoblast densities after only 7 days in culture. Again, the difference can be attributed to the seeding density variance between studies. The primary limitation of this culture system is the depth of cell growth. Histological sections revealed considerable cell proliferation and mineralized tissue formation within approximately 120–250 mm of the polymer foam surface, with only a minimal number of cells located in the center. This might be due to diffu- sion limitations of the media into the center of the foams because of distance and physical obstruction by cells and mineralized matrix present on the upper polymer surfaces. This problem may be alleviated by alterations in culture conditions or seeding methods, such as culturing the osteoblast foam constructs under mixed conditions31 or seeding the cells into the center of the foam rather than onto the surface, eliminating the possibility of surface obstruction to diffusion. For in vivo applications, however, diffusion limitations eventually may be overcome by the ingrowth of vas- cular tissue if viability is maintained until neovascu- larization is complete.9 The mineralized volume per surface area increased more dramatically than the penetration depth of mineralized tissue. This suggests that as long as the metabolic needs of the osteoblast are met, as they are on the surface, they will continue to lay down new osteoid tissue. CONCLUSIONS This study has demonstrated that rat stromal osteo- blasts can be cultured on 3-D porous poly(lactic-co- glycolic acid) foams to form a calcified bone-like tissue in vitro. Osteoblast proliferation and function were not affected by polymer foam pore size in the range of 150–710 mm and increased over time for all constructs. Cell seeding density affected initial osteoblast attach- ment and proliferation rate, but differences became less significant over time, with no measurable differ- ence in function. 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