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Tissue Engineering - John P. Fisher

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in small animals, then a well-controlled study in large animals such as primates may be considered before human clinical trials.

In this chapter, we first discuss the general considerations when selecting an animal model and the most commonly used animals in orthopedic research. Then, specific animal models for testing the biocompatibility and biodegradation of tissue-engineered constructs are described. Well-established animal models for the evaluation of osteogeneic or chondrogenic potential of these constructs are introduced. Finally, the experimental design and evaluation methods involved in the animal studies are reviewed.

16.2 Animal Model Selection

Studies using animal models may be deemed necessary if there are no other in vitro alternatives, the knowledge gained can be applied for the benefit of humans or animals, and the procedure does not cause extreme pain or disability to the animals. In an ideal animal model, the anatomy and physiology should be suitable for the specific study design, the pathogenesis and disease progression should parallel that of humans, and there should be a similar histopathologic response to that seen in humans [1]. The preclinical animal models in which the tissue-engineered constructs are tested should mimic the clinical situation as closely as possible.

The use of animals in tissue engineering research has become a scientific as well as an ethical issue. Generally, the use of invertebrates is preferred over vertebrates and the use of rats, rabbits, goats, and sheep are preferred over dogs and cats due to their pet status. All animal protocols should be approved by the researcher’s Institutional Animal Care and Use Committee to ensure that the experiments are appropriately designed, the number of animals is justified, and the animal procedures comply with the Animal Welfare Act.

From a practical perspective, animals of a particular species, strain, type, age, or weight should be easily available for the entire experimental study [1]. It is preferable that the local animal research facility has the capacity to house these animals. The animals should also be easy to handle in terms of transportation, housing, peri-operative care, specimen handling, and disposal. The susceptibility of animals to disease is also an important consideration especially for long-term survival studies. Finally, the costs of animal purchasing, transportation, time for quarantine, housing, surgical supply, and special equipment should be carefully calculated before the project begins. Generally, larger animals impose more housing and handling difficulties than small animals such as rats and rabbits, and are more expensive.

16.3 Commonly Used Animals

Although a lower level vertebrate, the rat is among the most popular animal subjects in tissue engineering orthopedic research and often the first to consider for a new study due to its low cost and easy care and handling. Rats have a mean healthy life span of 21 to 24 months. After bone elongation ceases by the age of 6 to 9 months, considerable useful lifespan remains for experimental evaluation [1]. Rats have been used extensively in studies of biocompatibility [2], fracture [3], and bone defect repair [4]. The mouse, also a small rodent, has gained popularity in tissue engineering research because its genome can be easily manipulated and investigated. Both regular and immunocompromised nude mice have been widely used for osteogenesis [5] and chondrogenesis [6] in subcutaneous tissue.

Rabbits are another commonly used animal in tissue engineering research. They are a relatively higher vertebrate, with an appropriate size for surgical operation and analysis. Rabbits are suitable for studies of bone defect repair [7], articular cartilage repair [8], ligament reconstruction [9], and spine fusion [10]. The dog, a higher-level vertebrate, has also played a dominant role in orthopedic research and has been extensively used in studies of bone defect repair [11], cartilage repair [12], ligament reconstruction [9], and meniscus repair [13].

Goats have become increasingly popular in tissue engineering research. They have been used for studies of biocompatibility [14], bone repair [15], cartilage repair [16], meniscus repair [17], ligament

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reconstruction [9], and spine fusion [18]. Sheep are large animals similar to goats, but less literature is available for their application in tissue engineering research. They have been used in studies of bone defect repair [19], cartilage defect repair [20], meniscus repair [13], and ligament reconstruction [9].

Pigs have been used in studies such as osteonecrosis of the femoral head [21] and fractures of cartilage and bone [22]. A miniature pig articular cartilage defect model has also been established [23]. Horses have been used mainly for the studies of cartilage and joint conditions due to their rich cartilage tissue [24]. Primates would be ideal for tissue engineering research because they are the closest to humans; however, due to lack of availability and high cost, they are only chosen when absolutely necessary as a step before human clinical trials. They have been used in studies of bone repair [25], cartilage repair [26], and spinal conditions [27].

Other animals in orthopedic research, though less frequently used, include hamsters for implant infection, chickens for scoliosis and tendon repair, turkeys for bone remodeling, emus for osteonecrosis of the femoral head, cats for osteoarticular transplantation, and guinea pigs for osteoarthritis [1].

16.4 Specific Animal Models

16.4.1 Biocompatibility

The biocompatibility study, as the first in vivo step of biomaterial evaluation, is typically performed by subcutaneous or intramuscular implantation in rats or rabbits. The rat is more economical and offers about a 15 month observation period, while the rabbit is used for longer-term evaluations of up to 6 months. Discs of 10 mm in diameter and 1 mm in thickness are commonly inserted in the dorsal subcutaneous tissue or back muscles (up to six implants per rat and eight to ten per rabbit) [28]. Alternatively, porous discs, biomaterials with seeded or encapsulated cells, and biomaterials contained in a stainless steel wire mesh cage have been studied [29,30].

The factors that determine the biocompatibility of a biomaterial include its chemistry, structure, morphology, and degradation. Biomaterial implantation often induces an inflammatory response through the activation of macrophages. The degree of fibrosis and vascularization of the tissue reaction dictate the nature of the response [29]. For instance, a mature fibrous capsule was observed after 12 weeks surrounding the implanted poly(propylene fumarate)/beta-tricalcium phosphate (PPF/beta-TCP) scaffolds in rats. Photocrosslinked PPF scaffolds also elicited a mild inflammatory response in rabbits [31]. The soft tissue response to oligo(poly(ethylene glycol)fumarate) (OPF) hydrogels was affected by the block length of poly(ethylene glycol) in a rabbit model [32]. In a granular tissue reaction, fibrovascular tissue ingrowth into porous poly(l-lactic acid) (PLLA) scaffolds was found to depend on the pore size [33].

A wide range of other biodegradable materials have also been assessed for their biocompatibility including commonly used poly(lactic acid) (PLA), poly(glycolic acid) (PGA), and their copolymers [34], as well as natural materials such as crosslinked gelatin [35] and hyaluronic acid [36].

16.4.2 Biodegradation

Biodegradable materials should be fully characterized for their degradation properties in vitro, but these studies cannot substitute for in vivo evaluation of degradation. Often, the rate of material degradation is significantly different in vivo compared to the in vitro condition. For example, poly(dl-lactic-co-glycolic acid) (PLGA) foams [37] and injectable PPF/beta-TCP composites [2] were both found to degrade more rapidly in vivo than in vitro. Differences in degradation rate in vivo vs. in vitro may be due to the extent of perfusion, pH levels, enzymatic or inflammatory mechanisms, or mechanical loading. Moreover, the apparent biocompatibility of a material may be confounded by greater toxicity of degradation products or material fragments.

Initial in vivo biodegradation studies may be undertaken in models similar or identical to those addressed in Section 16.4.1. Typically, discs, rods, foams, or other constructs are implanted in mice or rabbits and evaluated at multiple time intervals for changes in dry mass, molecular weight, mechanical

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properties, dimensions, and geometry. Standardization of sample geometry and dimensions is desirable to allow for comparison among research groups [28]. Samples may be implanted in various soft tissue locations, including the subdermal or subcutaneous space, intramuscular regions, or in the mesentery or intraperitoneal cavity.

Tests under loading conditions similar to the target tissue are ideal to include effects of mechanical loading on material degradation. If the material in question will have contact with bone, soft tissue evaluation must be followed or replaced by evaluation in bony tissue. Bone defect models are numerous [28], and generally involve drilled or otherwise excised bone segments. For example, self-reinforced PLLA and poly(dl-lactic acid) (PDLLA) [38] have been evaluated by implantation in mandibular bone or a femoral cavity of Sprague-Dawley rats. Because bone naturally remodels, it exhibits great healing capacity, and for this reason, critical sized defects are often necessary for long-term evaluation. These are defects that will predictably not heal spontaneously.

The choice of animal model to evaluate biodegradation in vivo also depends on the duration of the biodegradation study. For 1 to 6-month implantation studies, mice, rats, or rabbits can be used. Subdermal or subcutaneous implantation for up to 90 days in Sprague-Dawley rats has been used to evaluate biodegradation of poly(carbonate urethane), poly(ether urethane) [39], poly(ester amides) [40], poly(ether carbonates) [30], and poly(tetrahydrofuran) [41]. However, for long-term implantation studies, larger animals such as goats, sheep, or dogs should be used. As an example, sheep have been used as a model to evaluate biodegradation of self-reinforced poly(l-lactide) screws for up to 3 years in vivo [42,43].

16.4.3 Osteogenesis

In bone tissue engineering applications, after promising in vitro cell culture results are revealed, it is often a first step to use a heterotopic animal model for testing in vivo osteogenesis of the constructs. The major heterotopic models include the subcutaneous model, intramuscular model, intraperitoneal model, and mesentery model. Tissue-engineered constructs, either alone or placed in diffusion chambers can be implanted in mice, rats, rabbits, dogs, pigs, goats, or primates [44]. The rat subcutaneous model is the most frequently used. For example, bone formation was observed by marrow stromal osteoblast transplantation in a porous ceramic [45]. Ectopic bone formation was also demonstrated by transplantation of PLGA foams to the rat mesentery [46].

Although the heterotopic models are useful to provide some information helping to bridge the gap of in vitro and in vivo studies, the results obtained may not be the same once the constructs are implanted in actual bone defects. Therefore, further studies must be performed in bone defect models. Among them, the rabbit calvarial defect model is a very popular and reproducible bone defect model [44]. The calvarial bone is a plate which allows creation of a uniform defect that enables convenient radiographic and histological analysis. The calvarial bone has an appropriate size for easier surgical procedures and specimen handling. Because of the support by the dura and the overlying skin, no fixation is needed. This model has recently been used to evaluate the osteogenicity of a composite scaffold of PPF and PLGA [47], and growth-factor-coated PPF scaffolds [48].

Common long bone defect models include the rabbit radial model (most popular), rat femoral model, and the dog radial model. A composite bone scaffold consisting of nano-hydroxyapatite, collagen, and PLA was found to integrate the rabbit radial defect after 12 weeks [49]. The effect of bone morphogenetic protein was evaluated in the rat femoral defect model [4]. It should be noted that the rat model requires either internal or external fixation. In the dog model, ulnae fractures may occur, resulting in unexpected loss.

Due to the regenerative capability of bone defects, it is typical in tissue engineering research to consider critical sized defects. The critical size of the defect (CSD), defined as the smallest size that does not heal by itself if left untreated over a certain period of time, is 15 mm in diameter for adult New Zealand white rabbit calvarial defect model. The rat calvarial model is also popular with a CSD of 8 mm in diameter. A bone biomimetic device consisting of a porous biodegradable scaffold of poly(dl-lactide) and type I collagen, human osteoblast precursor cells, and rhBMP-2 was shown to promote bone regeneration in

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this model [50]. The CSD of long bones is at least two times the bone diameter (e.g., 12 to 15 mm for rabbit radial model and 5 to 10 mm for rat femoral model).

16.4.4 Chondrogenesis

Similar to bone tissue engineering, the first step to test the chondrogenic potential of tissue-engineered cartilage constructs in vivo is to use heterotopic models by subcutaneous, intramuscular, or intraperitoneal implantation in nude mice, syngeneic mice, rats, or rabbits. Among them, implantation of the constructs in dorsal subcutaneous pouches of nude mice is the most popular. For example, constructs containing genetically engineered chondrocytes were implanted into nude mice to demonstrate transgene expression and synthesis of glycosaminoglycan [51].

Commonly used cartilage defect models include partial-thickness (chondral) and full-thickness (osteochondral) defects in rabbits and dogs [52]. The rabbit distal femoral defect model is a well-established, reproducible model. Creating a partial-thickness defect can be challenging because the cartilage thickness in the rabbit femoral condyles is only 0.25 to 0.75 mm (compared to 2.2 to 2.4 mm in humans) [53]. The dog distal femur defect model, by contrast, offers a larger defect size with which to work. Besides species, the age of the animal is also an important consideration since the potential for cartilage repair as well as the response to various treatments varies with different animals [53]. Skeletal maturity of the commonly used New Zealand white rabbits typically occurs between 4 and 6 months.

Tissue-engineered articular cartilage is generally anchored to a defect by press fitting, suture, fibrin glue, or by using a periosteal flap. The local mechanical environment is well known to influence chondrogenesis and tissue healing. In the rabbit knee model, a defect created on the distal femoral surface is considered weight bearing, while a defect in the intercondylar groove is partial weight bearing [52]. The type of postoperative treatment should also be considered; the use of continuous passive motion can enhance cartilage healing [54] whereas joint immobilization may lead to decreased articular cartilage regeneration [55].

Many naturally derived and synthetic polymers are currently used as scaffolds for regeneration of articular cartilage [56]. They function not only as a vehicle for the delivery and retention of chondrogenic cells, but also as a substrate for cell attachment and matrix production. Natural polymeric materials such as collagen, hyaluronic acid, alginate, and fibrin glue have been extensively studied, as well as many synthetic polymers including PLA, PGA, and poly(ethylene oxide) (PEO) [53]. More recently, a new synthetic hydrogel based on oligopolyethylene glycol fumarate (OPF) was developed for cartilage tissue engineering [57].

16.5 Experimental Studies

16.5.1 Experimental Design

The experimental design includes the experimental groups to be used, sample size, sampling error, and control groups. The number of animals depends on the intrinsic variability among the animals, the consistency of the surgical procedure, the accuracy of the evaluation methods, and the choice of statistical method for data analysis. The number of animals needed in a study can often be determined from the results of a preliminary pilot study. For example, six to eight animals can be used in a preliminary study with histomorphometry or mechanical testing as the evaluation methods [58,59]. From this preliminary study, the standard deviation of the mean, coefficient of variation, and mean difference among the groups are determined. The sample size is affected by the desired power, the acceptable significance level, and the expected effect size. To reduce the interanimal variance, the animals should be of the same strain, sex, age, and weight. Some of the experimental animal models such as Sprague-Dawley rats and New Zealand white rabbits have identical genetic traits, but dogs and cats are relatively heterogeneous [60]. Therefore larger numbers of animals are often required for studies employing dogs or cats. The number of animals also depends on the evaluation methods.

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Sampling error reflects the inherent uncertainty of results about a population based on information gained from sample data, which is a subset of the population. Two sampling procedures used in biomedical research are random and stratified sampling [61,62]. In simple random sampling, each element of a population has an equal probability of being included. In stratified sampling, the elements are divided into nonoverlapping blocks, and specimens are chosen from each block by simple random sampling. Stratified sampling provides greater control over the distribution of specimens.

Controls in an experimental study include normal controls, treatment controls, and time controls. Unilateral, bilateral, unicortical, and bicortical bone models are being used in bone tissue engineering studies. Because of more efficient comparison between control and experimental groups, bilateral models are extensively used for evaluating biocompatibility and function of foreign materials in bones [63,64]. When major procedures are performed involving a joint, a unilateral model should be designed to allow the animal to heal over time. The bilateral cortical model is a design that doubles the number of specimens for testing if it is appropriate from animal use perspective [65,66].

16.5.2 Evaluation Methods

The evaluation method should be valid and capable of measuring the parameter of interest. The development of new evaluation methods requires assessment of its accuracy. Sources of error in an evaluation method are inadequate surgical procedures or specimen preparation, systemic error of testing systems, and data collection error. Clinical evaluation, necroscopy, morphological or structural analysis, biochemical evaluation, mechanical testing, and the use of specialized devices are used in orthopedic research for evaluation. The most commonly used methods in orthopedic animal research are clinical observation, radiography, histological evaluation, and mechanical testing [60].

One method of biocompatibility evaluation is the implantation of disk-shaped samples of the material into the right and left epididymal fat pads or the right and left rear haunch subcutaneous tissue [67]. The following are removed at autopsy: the implant with its surrounding capsule, skin and subcutaneous tissue, the axillary and popliteal lymph nodes, heart, kidneys, lungs, liver, spleen, brain, and aorta, as well as any macroscopically unusual findings in the stomach, bowels, and mesenteric lymph nodes [68,69]. For evaluation of bone growth in explanted skeletal specimens, the total cross-sectional area of newly formed trabecular bone is determined on sequential longitudinal sections [70–72]. Confocal microscopy is utilized for cell visualization and morphology [73]. Alkaline phosphatase activity and calcium content are measured to assess the extent of mineralization in newly formed bone [74,75]. Western analyses of the newly synthesized collagen types I, II, and IX from the explanted samples are done using monoclonal antibodies and chemiluminescent substrate [76,77].

The explanted bone samples should be tested intact to establish reference mechanical properties [78]. Loads should be applied in a manner that minimizes focal loading of the specimen, so that accurate, representative material properties are measured. Preconditioning the specimens by cyclic loading, over several complete load-unload cycles, to a peak compressive force of approximately one average body weight (70 kg) is reasonable to do. This technique tends to smooth out variability in the data, and increases the likelihood of obtaining reproducible data.

References

[1]An, Y.H. and Friedman, R.J., Animal selections in orthopaedic research, in Animal Models in Orthopaedic Research, An, Y.H. and Friedman, R.J. (Eds.), CRC Press, Boca Raton, FL, 1999, p. 39.

[2]Peter, S.J. et al., In vivo degradation of a poly(propylene fumarate)/beta-tricalcium phosphate injectable composite scaffold, J. Biomed. Mater. Res. 41, 1, 1998.

[3]An, Y. et al., Production of a standard closed fracture in the rat tibia, J. Orthop. Trauma 8, 111, 1994.

Animal Models for Orthopedic Implants

16-7

[4]Yasko, A.W. et al., The healing of segmental bone defects, induced by recombinant human bone morphogenetic protein (rhBMP-2). A radiographic, histological, and biomechanical study in rats,

J. Bone Joint Surg. Am. 74, 659, 1992.

[5]Miyazawa, K., Kawai, T., and Urist, M.R., Bone morphogenetic protein-induced heterotopic bone in osteopetrosis, Clin. Orthop. Relat. Res. 324, 259, 1996.

[6]Paige, K.T. et al., De novo cartilage generation using calcium alginate-chondrocyte constructs, Plast. Reconstr. Surg. 97, 168, 1996.

[7]Roy, T.D. et al., Performance of degradable composite bone repair products made via threedimensional fabrication techniques, J. Biomed. Mater. Res. 66A, 283, 2003.

[8]Wakitani, S. et al., Repair of large full-thickness articular cartilage defects with allograft articular chondrocytes embedded in a collagen gel, Tissue Eng. 4, 429, 1998.

[9]Carpenter, J.E. and Hankenson, K.D., Animal models of tendon and ligament injuries for tissue engineering applications, Biomaterials 25, 1715, 2004.

[10]Boden, S.D., Biology of lumbar spine fusion and use of bone graft substitutes: present, future, and next generation, Tissue Eng. 6, 383, 2000.

[11]Johnson, K.D. et al., Evaluation of ground cortical autograft as a bone graft material in a new canine bilateral segmental long bone defect model, J. Orthop. Trauma 10, 28, 1996.

[12]Lee, C.R. et al., Effects of a cultured autologous chondrocyte-seeded type II collagen scaffold on the healing of a chondral defect in a canine model, J. Orthop. Res. 21, 272, 2003.

[13]Buma, P. et al., Tissue engineering of the meniscus, Biomaterials 25, 1523, 2004.

[14]Mendes, S.C. et al., Biocompatibility testing of novel starch-based materials with potential application in orthopaedic surgery: a preliminary study, Biomaterials 22, 2057, 2001.

[15]Kruyt, M.C. et al., Optimization of bone–tissue engineering in goats, J. Biomed. Mater. Res. 69B, 113, 2004.

[16]Butnariu-Ephrat, M. et al., Resurfacing of goat articular cartilage by chondrocytes derived from bone marrow, Clin. Orthop. Relat. Res. 330, 234, 1996.

[17]Port, J. et al., Meniscal repair supplemented with exogenous fibrin clot and autogenous cultured marrow cells in the goat model, Am. J. Sports Med. 24, 547, 1996.

[18]Kruyt, M.C. et al., Bone tissue engineering and spinal fusion: the potential of hybrid constructs by combining osteoprogenitor cells and scaffolds, Biomaterials 25, 1463, 2004.

[19]Viljanen, V.V. et al., Xenogeneic mouse (Alces alces) bone morphogenetic protein (mBMP)-induced repair of critical-size skull defects in sheep, Int. J. Oral Maxillofac. Surg. 25, 217, 1996.

[20]Homminga, G.N. et al., Repair of sheep articular cartilage defects with a rabbit costal perichondrial graft, Acta Orthop. Scand. 62, 415, 1991.

[21]Seiler, J.G., III et al., Posttraumatic osteonecrosis in a swine model. Correlation of blood cell flux, MRI and histology, Acta Orthop. Scand. 67, 249, 1996.

[22]Tomatsu, T. et al., Experimentally produced fractures of articular cartilage and bone. The effects of shear forces on the pig knee, J. Bone Joint Surg. Br. 74, 457, 1992.

[23]Hunziker, E.B., Driesang, I.M., and Morris, E.A., Chondrogenesis in cartilage repair is induced by members of the transforming growth factor-beta superfamily, Clin. Orthop. Relat. Res. 391S, S171, 2001.

[24]Litzke, L.E. et al., Repair of extensive articular cartilage defects in horses by autologous chondrocyte transplantation, Ann. Biomed. Eng. 32, 57, 2004.

[25]Cancian, D.C. et al., Utilization of autogenous bone, bioactive glasses, and calcium phosphate cement in surgical mandibular bone defects in Cebus apella monkeys, Int. J. Oral Maxillofac. Implants 19, 73, 2004.

[26]Buckwalter, J.A. et al., Osteochondral repair of primate knee femoral and patellar articular surfaces: implications for preventing post-traumatic osteoarthritis, Iowa Orthop. J. 23, 66, 2003.

[27]Boden, S.D., Grob, D., and Damien, C., Ne-osteo bone growth factor for posterolateral lumbar spine fusion: results from a nonhuman primate study and a prospective human clinical pilot study, Spine 29, 504, 2004.

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[28]An, Y.H. and Friedman, R.J., Animal models for testing bioabsorbable materials, in Animal Models in Orthopaedic Research, An, Y.H. and Friedman, R.J. (Eds.), CRC Press, Boca Raton, FL, 1999, p. 219.

[29]Babensee, J.E. et al., Host response to tissue engineered devices, Adv. Drug Deliv. Rev. 33, 111, 1998.

[30]Dadsetan, M. et al., In vivo biocompatibility and biodegradation of poly(ethylene carbonate),

J. Control. Release 93, 259, 2003.

[31]Fisher, J.P. et al., Soft and hard tissue response to photocrosslinked poly(propylene fumarate) scaffolds in a rabbit model, J. Biomed. Mater. Res. 59, 547, 2002.

[32]Shin, H. et al., In vivo bone and soft tissue response to injectable, biodegradable oligo(poly(ethylene glycol) fumarate) hydrogels, Biomaterials 24, 3201, 2003.

[33]Wake, M.C., Patrick, C.W., Jr., and Mikos, A.G., Pore morphology effects on the fibrovascular tissue growth in porous polymer substrates, Cell Transplant 3, 339, 1994.

[34]Behravesh, E. et al., Synthetic biodegradable polymers for orthopaedic applications, Clin. Orthop. Relat. Res. 367S, S118, 1999.

[35]Hong, S.R. et al., Biocompatibility and biodegradation of cross-linked gelatin/hyaluronic acid sponge in rat subcutaneous tissue, J. Biomater. Sci. Polym. Ed. 15, 201, 2004.

[36]Baier Leach, J. et al., Photocrosslinked hyaluronic acid hydrogels: natural, biodegradable tissue engineering scaffolds, Biotechnol. Bioeng. 82, 578, 2003.

[37]Lu, L. et al., In vitro and in vivo degradation of porous poly(dl-lactic-co-glycolic acid) foams, Biomaterials 21, 1837, 2000.

[38]Majola, A. et al., Absorption, biocompatibility, and fixation properties of polylactic acid in bone tissue: an experimental study in rats, Clin. Orthop. Relat. Res. 268, 260, 1991.

[39]Christenson, E.M. et al., Poly(carbonate urethane) and poly(ether urethane) biodegradation: in vivo studies, J. Biomed. Mater. Res. 69A, 407, 2004.

[40]Tsitlanadze, G. et al., Biodegradation of amino-acid-based poly(ester amide)s: in vitro weight loss and preliminary in vivo studies, J. Biomater. Sci. Polym. Ed. 15, 1, 2004.

[41]Pol, B.J. et al., In vivo testing of crosslinked polyethers. I. Tissue reactions and biodegradation,

J. Biomed. Mater. Res. 32, 307, 1996.

[42]Jukkala-Partio, K. et al., Biodegradation and strength retention of poly-l-lactide screws in vivo. An experimental long-term study in sheep, Ann. Chir. Gynaecol. 90, 219, 2001.

[43]Suuronen, R. et al., A 5-year in vitro and in vivo study of the biodegradation of polylactide plates,

J. Oral Maxillofac. Surg. 56, 604, 1998.

[44]An, Y.H. and Friedman, R.J., Animal models of bone defect repair, in Animal Models in Orthopaedic Research, An, Y.H. and Friedman, R.J. (Eds.), CRC Press, Boca Raton, FL, 1999, p. 241.

[45]Gao, J. et al., Tissue-engineered fabrication of an osteochondral composite graft using rat bone marrow-derived mesenchymal stem cells, Tissue Eng. 7, 363, 2001.

[46]Ishaug-Riley, S.L. et al., Ectopic bone formation by marrow stromal osteoblast transplantation using poly(dl-lactic-co-glycolic acid) foams implanted into the rat mesentery, J. Biomed. Mater. Res. 36, 1, 1997.

[47]Dean, D. et al., Poly(propylene fumarate) and poly(dl-lactic-co-glycolic acid) as scaffold materials for solid and foam-coated composite tissue-engineered constructs for cranial reconstruction, Tissue Eng. 9, 495, 2003.

[48]Vehof, J.W. et al., Bone formation in transforming growth factor beta-1-coated porous poly(propylene fumarate) scaffolds, J. Biomed. Mater. Res. 60, 241, 2002.

[49]Liao, S.S. et al., Hierarchically biomimetic bone scaffold materials: nano-HA/collagen/PLA composite, J. Biomed. Mater. Res. 69B, 158, 2004.

[50]Winn, S.R. et al., Tissue-engineered bone biomimetic to regenerate calvarial critical-sized defects in athymic rats, J. Biomed. Mater. Res. 45, 414, 1999.

[51]Madry, H. et al., Gene transfer of a human insulin-like growth factor I cDNA enhances tissue engineering of cartilage, Hum. Gene Ther. 13, 1621, 2002.

Animal Models for Orthopedic Implants

16-9

[52]An, Y.H. and Friedman, R.J., Animal models of articular cartilage defect, in Animal Models in Orthopaedic Research, An, Y.H. and Friedman, R.J. (Eds.), CRC Press, Boca Raton, FL, 1999, p. 309.

[53]Reinholz, G.G. et al., Animal models for cartilage reconstruction, Biomaterials 25, 1511, 2004.

[54]O’Driscoll, S.W. and Salter, R.B., The repair of major osteochondral defects in joint surfaces by neochondrogenesis with autogenous osteoperiosteal grafts stimulated by continuous passive motion. An experimental investigation in the rabbit, Clin. Orthop. Relat. Res. 208, 131, 1986.

[55]Vanwanseele, B., Lucchinetti, E., and Stussi, E., The effects of immobilization on the characteristics of articular cartilage: current concepts and future directions, Osteoarthr. Cartil. 10, 408, 2002.

[56]Temenoff, J.S. and Mikos, A.G., Injectable biodegradable materials for orthopedic tissue engineering, Biomaterials 21, 2405, 2000.

[57]Holland, T.A. et al., Transforming growth factor-beta1 release from oligo(poly(ethylene glycol) fumarate) hydrogels in conditions that model the cartilage wound healing environment, J. Control Release 94, 101, 2004.

[58]Munro, B.H., Jacobson, B.J., and Braitman, L.E., Introduction to inferential statistics and hypothesis testing, in Statistical Methods for Health Care Research, 2nd ed., Munro, B.H. and Page, E.B. (Eds.), Lippincott, Philadelphia, 1993.

[59]Matthews, D.E. and Farewell, V.T., Using and Understanding Medical Statistics, 3rd ed, Basel Karger, London, 1996.

[60]An, Y.H. and Bell, T.D., Experimental design, evaluation methods, data analysis, publication, and research ethics, in Animal Models in Orthopaedic Research, An, Y.H. and Friedman, R.J. (Eds.), CRC Press, Boca Raton, FL, 1999, p. 15.

[61]Manly, B.J., The Design and Analysis of Research Studies, Cambridge University Press, Cambridge, 1992.

[62]Forthofer, R.N. and Lee, E.S., Introduction to Biostatistics. A Guide to Design, Analysis, and Discovery, Academic Press, San Diego, CA, 1995.

[63]Laberge, M. and Powers, D.L., Scientific basis for bilateral animal models in orthopaedics, J. Invest. Surg. 4, 109, 1991.

[64]An, Y.H. et al., Fixation of osteotomies using bioabsorbable screws in the canine femur, Clin. Orthop. Relat. Res. 355, 300, 1998.

[65]Thomas, K.A. and Cook, S.D., An evaluation of variables influencing implant fixation by direct bone apposition, J. Biomed. Mater. Res. 19, 875, 1985.

[66]Thomas, K.A. et al., The effect of surface treatments on the interface mechanics of LTI pyrolytic carbon implants, J. Biomed. Mater. Res. 19, 145, 1985.

[67]Kidd, K.R. et al., A comparative evaluation of the tissue responses associated with polymeric implants in the rat and mouse, J. Biomed. Mater. Res. 59, 682, 2002.

[68]Nary Filho, H. et al., Comparative study of tissue response to polyglecaprone 25, polyglactin 910 and polytetrafluorethylene structure materials in rats, Braz. Dent. J. 13, 86, 2002.

[69]Eltze, E. et al., Influence of local complications on capsule formation around model implants in a rat model, J. Biomed. Mater. Res. 64A, 12, 2003.

[70]Lewandrowski, K.U. et al., Effect of a poly(propylene fumarate) foaming cement on the healing of bone defects, Tissue Eng. 5, 305, 1999.

[71]Schantz, J.T. et al., Induction of ectopic bone formation by using human periosteal cells in combination with a novel scaffold technology, Cell Transplant 11, 125, 2002.

[72]Yasin, M. and Tighe, B.J., Polymers for biodegradable medical devices. VIII. Hydroxybutyratehydroxyvalerate copolymers: physical and degradative properties of blends with polycaprolactone,

Biomaterials 13, 9, 1992.

[73]Behravesh, E. and Mikos, A.G., Three-dimensional culture of differentiating marrow stromal osteoblasts in biomimetic poly(propylene fumarate-co-ethylene glycol)-based macroporous hydrogels,

J. Biomed. Mater. Res. 66A, 698, 2003.

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Tissue Engineering

[74]Temenoff, J.S. et al., In vitro osteogenic differentiation of marrow stromal cells encapsulated in biodegradable hydrogels, J. Biomed. Mater. Res. 70A, 235, 2004.

[75]Temenoff, J.S. et al., Thermally cross-linked oligo(poly(ethylene glycol) fumarate) hydrogels support osteogenic differentiation of encapsulated marrow stromal cells in vitro, Biomacromolecules 5, 5, 2004.

[76]Adkisson, H.D. et al., In vitro generation of scaffold independent neocartilage, Clin. Orthop. Relat. Res. 391S, S280, 2001.

[77]Gibson, G. et al., Type X collagen is colocalized with a proteoglycan epitope to form distinct morphological structures in bovine growth cartilage, Bone 19, 307, 1996.

[78]Elder, S. et al., Biomechanical evaluation of calcium phosphate cement-augmented fixation of unstable intertrochanteric fractures, J. Orthop. Trauma 14, 386, 2000.

17

The Regulation of

Engineered Tissues:

Emerging Approaches

17.1 Introduction.............................................. 17-1

17.2 FDA Regulation .......................................... 17-2

Classification of Medical Products Special Product

Designations Human Cellular and Tissue-Based Products

Marketing Review and Approval Pathways

17.3Regulation of Pharmaceutical/Medical Human

Tissue Products in Europe .............................. 17-10 17.4 Regulation of Pharmaceutical/Medical Human

Tissue Products in Japan ................................ 17-11 17.5 Other Considerations Relevant to Engineered

Kiki B. Hellman Tissues .................................................... 17-12

The Hellman Group, LLC

FDA Regulation and Product Liability Ownership of

Human Tissues

David Smith

17.6 Conclusion ...............................................

17-14

Teregenics, LLC

References .......................................................

17-14

17.1 Introduction

A critical step in translating tissue engineering research into product applications for the clinic and marketplace is understanding the strategies developed by government agencies for providing appropriate regulatory oversight. Since the eventual goal is the establishment of a global industry with the ability of companies to market products across national boundaries, a harmonized international regulatory approach would be ideal. However, while groups actively work toward that end, and, recognizing that market acceptance can be influenced by local cultural, ethical, and legal concerns, it is essential to recognize and appreciate the approaches of the regulatory agencies of those countries where engineered tissue research is already moving into product development and clinical application. While the science in the field is now worldwide in scope [1], we will limit our discussion to the regulatory approaches of the United States, with attention to emerging trends in Europe and Japan.

The U.S. Food and Drug Administration (FDA) has recognized that an important segment of the products it regulates arises from applications of new technology such as tissue engineering, and that,

17-1