ORTHOPEDICS

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BASIC AVIAN BONE GROWTH AND HEALING

Thomas N. Tully, Jr., DVM, MS, Dipl ABVPAvian

Orthopedic injuries (ranging from trauma-induced periosteal inammation to open, severely comminuted long-bone fractures) commonly occur in the many different avian species that are treated by veterinarians. When assessing these injuries, the veterinarian must understand basic avian bone anatomy and healing to provide adequate treatment. The knowledge of avian bone structure anatomy aids the determination of not only treatment decisions but also the prognosis for healing and function. These skills are important when being consulted by a variety of avian stewards, including the companion bird owner who wants to know if their pet will be able to perch or copulate properly and the wildlife rehabilitator who wants to know if the bird can be released. This article, although basic in nature, provides information that every veterinarian should know to treat avian bone injuries properly and to communicate the prognosis and current status of those injuries to owners and other interested parties. The structural makeup of bone tissue is similar across all animal species. Generalizing the mechanisms of bone growth and turnover for avian species with that of mammalian species (described in other articles of this text) is appropriate.13 This article focuses on avian specics of bone structure anatomy, while using basic anatomical information to serve as a foundation.

From the Departments of Veterinary Clinical Sciences, Louisiana State University of Veterinary Medicine, Baton Rouge, Louisiana

VETERINARY CLINICS OF NORTH AMERICA: EXOTIC ANIMAL PRACTICE

VOLUME 5 NUMBER 1 JANUARY 2002

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BASIC BONE ANATOMY In avian species, bone serves two major functions: as a support for the musculature and as a reservoir for calcium and phosphorus.13 This reservoir must be readily available to hens that lay eggs, and it may come from medullary bone, because the egg shell can contain up to 10% of the hens body stores of calcium.13 Bone primarily consists of cortical bone found in the shafts of long bones and cancellous bone, which are lightweight structure supports.13 The mineral part of bone provides compression resistance, whereas the organic ber supports resist bending and twisting forces.13

BONE FORMATION Avian long bones are developed by endochondral ossication. The long bone cells become chondrocytes that secrete extracellular matrix.13 The external cells develop into a perichondral sheath that consists of an outer layer of broblast that is rich with connective tissue and a multiplying inner layer of mesenchymal cells.13 These cells hypertrophy over time, accumulating glycogen and producing additional extracellular matrix.13 There is a simultaneous development of bone vascularity and cell differentiation of the perichondral cell line to osteolysis that secrete a bone collar.13 This collar gives rise to the calcied bone by osteoblastic expansion. At the center of this expanding collar, the chondrocytic secretion of alkaline phosphatase allows calcication to occur.13 The osteoclast cell line erodes the calcied cartilage. Areas formed by osteoclastic erosion ll with cells that form marrow tissue and osteoblasts.13 Periosteal bone is formed by an ever-expanding bone collar, whereas the marrow cavity is established as the internal trabecula formations are removed.13 Flat bones are formed from a mass of embryonic mesenchyme that gives rise to osteoblasts and a vasculature that sustain bone development.24 In each avian at bone, there are usually two of these embryonic mesenchymal development centers.13 The growth plates of avian and mammalian species are structurally distinct, but the mechanisms that control the physiologic processes are the same.26, 28 There is a difference in the vasculature of the growth plate between mammalian and avian species. Although the growth plates of rats have been shown to be avascular, avian epiphyseal vessels supply the transitional zone and metaphyseal vessels.10, 26 Mineralization of the growth plate depends on the extracellular matrix that surrounds the hypertrophic chondrocytes, similar to what occurs with long bone formation.13 Alkaline phosphatase (ALP) is essential for the growth-plate mineralization process to take place, whereas inhibition of ALP disrupts the calcication process.19, 20 Although only active ALP seems to be present in the calcication process, inactive ALP

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is present in all growth-plate zones, which indicates the need for active ALP to be present in the mineralization of the growth plate.2, 6 In avian species, the main regulator of post-hatch bone growth is growth hormone.13 Generally, the effect of growth hormone on developing bone is thought to occur via indirect increases in the production of hepatic insulin-like growth factor 1 (IGF-1). Receptors for IGF-1 are found predominantly on proliferating chondrocytes but the greatest concentration of these receptors are on the stem cell receptors.3, 11, 22, 27 In addition to its effect on proliferating chondrocytes, IGF-1 also stimulates the matrix synthesis of collagen and proteoglycans.9 Other agents that participate in the development of the growth plate include broblast growth factor and transforming growth factor , both of which synergistically stimulate chondrocyte proliferation and differentiation.5, 23

BONE REMODELING After initial bone growth ceases, bone must maintain its health and normal anatomical size and shape and must contribute to the maintenance of calcium homeostatsis.13 To accomplish these physiologic processes, certain cell types (e.g., osteoblasts, osteocytes) are required. Osteoblasts are members of the broblast-like cell group that comprises not only the bone cells but also chondrocytes, adipocytes, and myoblasts.16 As a single layer of cells on the bone surface, osteoblasts are columnar and highly packed in areas of high matrix formation and are compacted in areas of quiescence.18 In the active development of bone, osteoblasts produce an amount of unmineralized matrix (osteoid or type I collagen) that is related closely to the anatomical size of the cell.13 A diverse group of materials comprise the matrix of osteoid and are found in proportions that depend on the animal species and age and on the site of bone.1, 25 Collagen, a protein, is the major component of bone, comprising up to 90% of the organic content.1 This osteoid becomes mineralized, but there is always a thin layer of unmineralized matrix that separates the osteoblast cells from the bone. Other osteoblastic activities include regulating osteoclastic bone resorption and becoming quiescent.21 As the primary cell type that controls bone resorption, osteoblasts have receptors for the major systemic bone-resorptive agents.14 The resorptive agents trigger osteoblasts to summon osteoclasts to increase bone-resorption activity.13 Additionally, matrix-degrading enzymes produced by the osteoblasts may contribute to the initiation of bone resorption.13 Within the matrix of the bone are osteocytes, which may aid in calcium homeostasis by affecting the dissolution of bone mineral.4 The resorbing cells in large bones (during both the mineral and organic phases of bone) are called osteoclasts. These cells resorb bone during skeletal remodeling and can penetrate 50 to 70 m into compact bone in 24 hours.13 The amount of bone that osteoclasts can destroy in

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24 hours is equivalent to the amount of bone formed by 100 to 1000 osteoblast cells.13 There are only a few bone cell types (e.g., osteoblasts, osteoclasts) that are involved in most bone tissue remodeling. The osteoblast cell line helps with tissue development, whereas the osteoclast cell line helps with tissue resorption as mentioned previously. Systemic hormonal factors that affect resorption through the targeting of osteoblasts include parathyroid hormone; parathyroid-like peptide; and 1,25(OH)2D3 (the principal active metabolite of vitamin D3). During fracture repair in chickens, 1,25(OH)2D3 plasma levels have been found to be decreased, whereas 1,25(OH)2D3 and 24,25(OH)2D3 were increased in the callus tissue.12 Hormones that inhibit resorption by targeting osteoclasts are calcitonin and the calcitonin-gene-related protein. Estrogens, glucocorticoids, and retinoids also play a role in bone remodeling.17 Estrogentreated birds have shown reduced callus strength and an increased diameter of the callus owing to an instability of the fracture site, which demonstrates the effect of this hormone on avian bone repair.7 When bone formation and resorption take place synergistically under normal physiologic circumstances, it is called coupling.17 When pathology is involved with this formation-to-resorption ratio (e.g., osteoporosis), the process is termed uncoupling.17

CALCIUM AND PHOSPHORUS HOMEOSTASIS Bone structure not only provides support to the avian body but also stores calcium for the important physiologic processes that occur during egg formation. The most important calcium reservoir for egg development is the medullary bone reserve.1 Bone is a unique structure in the body because it is a composite structure that not only must be stable but also must provide calcium and phosphorus when necessary for metabolic balance. Although the conguration of bone is controlled genetically, it can be affected by internal and external inuences on the thickness of cortical bone, the amount of trabecular or cancellous bone, and the amount of membranous bone in the skull.

BONE STRUCTURE Collagen, a highly structured and organized protein, is a major component of bone, cartilage, skin, tendons, and ligaments. Twelve different forms of collagen have been identied in various body tissues, ranging from types I, II, III, V and IX (major bril forming) to types VI, VII and X (short chain). Bone collagen is incredibly strong: A single 1mm ber of this protein can support a weight of up to 40 kg.1 Where collagen is impregnated with hydroxyapatite microcrystals, it develops

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a highly compressive stiffness, which provides its structural strength and load-bearing properties.1 Collagen bers vary in diameter depending on the species and the area of bone within the body; as the animal ages, the bers often become packed more tightly and increase in diameter.27 Type I collagen is the major component of bone, but it also contains types V and XII.1 Cartilage is composed mostly of type II collagen.1 When collagen is formed, the hydroxylation of specic amino acids occurs in the presence of ascorbic acid.1 A deciency of ascorbic acid in the diet causes defective collagen to be formed, which is diagnosed clinically as scurvy in humans and guinea pigs. When collagen is formed correctly with all of the necessary ingredients, osteoid (composed mainly of collagen bers) transforms into fully mineralized bone.1 Within 1 to 2 weeks, collagen in the woven bone of an adult chickens bone defect is type I collagen that contains three times as much hydroxylysine as that normally found in adult avian bone.8 After the repaired bone reaches 8 weeks of age, the hydroxylysine is back to normal levels.8 Hydroxylysine-rich type I collagen can be manufactured by the osteoblasts of adult birds and young growing animals.8 Matrix vesicles (i.e., extracellular, membrane-bound particles that are formed from chondrocyte cell processes) are essential for the initial mineralization of the osteoid.1 During primary matrix vesicle calcication, there are three stages that have been associated with the calcication of epiphyseal cartilage in mammalian species: (1) formation of matrix vesicles and crystal needles, (2) mineral nodule formation, and (3) alignment of crystal with collagen brils.1 Matrix vesicles also may have a similar effect on avian bone formation.

AVIAN FRACTURE HEALING There have been limited studies on determining the physiologic and anatomical properties of avian fracture repair.15, 29 Investigations of avian fracture repair have used wing bones to determine tissue morphology during repair, histomorphometric and angiographic analysis of bone healing, and histologic results of avian bone repair using various repair methods.8, 15, 29 The amount of time needed for an avian fracture to heal decreases proportional to the amount of immobilization obtained at the fracture site.15 Newton and Zietlin15 showed that unilateral fractures of the radius and ulna developed a union of both bones by radiographic evidence of an mineralized external callus at 5 weeks after internal xation and at 8 weeks after external xation. Variations in the study by Newton and Zietlin15 also investigated various healing responses to different fractures by using external and internal xation techniques and the various complications by using these immobilization methods. When the radius had been fractured and the ulna kept intact, an endosteal cancellous callus was noted at 3 weeks;

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palpable motion was not evident when using external xation.15 Similar results were found when the ulna and radius had been fractured, with the ulna immobilized with a Kirschner wire and the radius remaining fractured.15 Interestingly, the healing of the immobilized ulna lagged behind that of the radius, because the radius union was primarily cortical bone and the ulna union was cancellous bone.15 When both bones were immobilized internally, the healing process was shortened; by week 4, the fractures to the pigeon ulna and radius had complete unions and had begun to remodel.15 This study showed that, although the internal healing process was accelerated with the use of internal xation, the function of the wing after repair was better with birds in which external xation was used.26 External xation does not interfere with soft tissue structures or joint spaces in any way that may impair future limb function. These results emphasize the critical nature of (1) using proper surgical techniques to implant internal xation devices, and (2) minimizing trauma to the surrounding soft tissue and joint spaces. Segmental fractures were found to heal best if the fragment was attached to tissue with an adequate blood supply.15 This blood supply is needed to nourish the periosteal surface, which has been shown to incorporate the largest amount of a healing callus tissue.29 Although the periosteal surface provides the largest amount of callus formation, the endosteal surface also is active during the healing process.29 The magnitude of periosteal surface involvement in callus formation may preclude the need for reformation of intramedullary circulation to achieve an osseous union in the bones of some avian species.29 SUMMARY There have been few studies on the process of fracture repair in avian species. Most of the information shows similarities between avian and mammalian bone growth and fracture repair, but there are differences. The main nding conrms that fractures must be reduced properly, stabilized, and immobilized with an adequate blood supply to the bone fragments for optimal healing. The return to function of extremities, particularly the legs and wings, is an important consideration when internal xation methods are used. Causing little or no collateral damage to soft tissue and joint when implanting internal hardware is ideal and reduces the likelihood of impaired function. Whether internal or external xation methods are used for fracture reduction, the knowledge of avian bone growth and fracture repair is essential for veterinarian understanding and when discussing the healing process with clients. References
1. Ali SY: Matrix formation and mineralization in bone. In Whitehead CC (ed): Bone Biology and Skeletal Disorders in Poultry: Poultry Science Symposium No. 23. Oxfordshire, England, Carafax Publishing, 1993, pp 1938

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2. Anderson HC: Biology of disease: Mechanisms of mineral formation in bone. Lab Invest 60:320330, 1989 3. Barnard R, Haynes KM, Wirther GA, et al: The ontogeny of growth hormone receptors in the rabbit tibia. Endocrinology 122:25622569, 1988 4. Bonucci E: The histology, histochemistry, and ultrastructure of bone. In Pecile A, de Bernard B (eds): Bone Regulatory Factors: Morphology, Biochemistry, Physiology, and Pharmacology. New York, Plenum, 1990, pp 15138 5. Crabb ID, OKeefe RJ, Puzas JE, et al: Synergistic effect of transforming growth factor and broblast growth factor on DNA synthesis in chick growth-plate chondrocytes. J Bone Miner Res 5:11051112, 1990 6. de Bernard B, Bianco P, Bonucci E, et al: Biochemical and immunohistochemical evidence that in cartilage an alkaline phosphatase is a Ca2-binding glycoprotein. J Cell Biol 103:16151623, 1986 7. Engin AE, Toney LR, Negulesco JA. Effects of oestrogen upon tensile properties of healing fracture avian bone. J Biomed 5:4954, 1983 8. Glimcher MJ, Shapiro F, Ellis RD, et al: Changes in tissue morphology and collagen composition during the repair of cortical bone in the adult chicken. Am J Bone Joint Surg 62:964973, 1980 9. Guenther HL, Guenther HE, Froesch ER, et al: Effect of insulin-like growth factor on collagen and glycosaminoglycan synthesis by rabbit articular chondrocytes in culture. experentia 979981, 1982 10. Hunter WL, Arsenault L: Vascular invasion of the epiphyseal growth plate: Analysis of metaphyseal capillary ultrastructure and growth dynamics. Anat Rec 227:223231, 1990 11. Isaksson OGP, Lindahl A, Nilsson A, et al: Mechanisms of the stimulatory effect of growth hormone on longitudinal bone growth. Endocr Rev 8:426439, 1987 12. Lidor C, Dekel S, Edelstein S: The metabolism of vitamin D3 during fracture healing in chicks. Endocrinology 120:389393, 1987 13. Loveridge N, Thomson BM, Farquharson C: In Whitehead CC (ed): Bone Biology and Skeletal Disorders in Poultry: Poultry Science Symposium No. 23. Oxfordshire, England, Carafax Publishing, 1993, pp 317 14. Marks SC, Popoff SN: Bone cell biology: The regulation of development, structure, and function in the skeleton. Am J Anatomy 183:144, 1988 15. Newton CD, Zeitlin S: Avian fracture healing. J Am Vet Med Assoc 170:620625, 1977 16. Owen M: Marrow stromal stem cells. J Cell Sci Suppl 10:6376, 1988 17. Price JS, Russell GG: In Whitehead CC (ed): Bone Biology and Skeletal Disorders in Poultry: Poultry Science Symposium No. 23. Oxfordshire, England, Carafax Publishing, 1993, pp 3960 18. Puzas JE: The osteoblast. In Farus MJ (ed): Primer on Metabolic Bone Disease and Disorders of Mineral Metabolism. Philadelphia, Lippincott, Williams and Wilkins, 1990, pp 1115 19. Register TC, Wuthier RE: Effect of L- and D-tetramisole on 32Pi and 45Ca uptake and mineralization by matrix-enriched factions from chicken epiphyseal cartilage. J Biol Chem 259:922928, 1984 20. Robinson R: The possible signicance of hexose phosphoric esters in ossication. Biochem J 17:286293, 1923 21. Rodan GA, Martin J: Role of osteoblasts in hormonal control of bone resorption: A hypothesis. Calcif Tissue Int 3:349351, 1981 22. Salmon WD, Daughaday WH: A hormonally controlled serum factor which stimulates sulfate incorporation by cartilage in vitro. J Lab Clin Med 49:825836, 1957 23. Schoeld JN, Wolpert L: Effect of TGF-1, TGF-2 and FGF on cartilage and muscle differentiation. Exp Cell Res 191:144148, 1990 24. Thompson TJ, Owens PH, Wilson DJ: Intramembranous osteogenesis and angiogenesis in the chick embryo. J Anat 166:5565, 1989 25. Triftt JT: The organic matrix of bone tissue. In Urist MR (ed): Fundamental and Clinical Bone Physiology. Philadelphia, Lippincott, 1980, pp 4582 26. Trueta J, Morgan JD: The vascular contribution to osteogenesis. J Bone Joint Surg Br 42:97109, 1960

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27. Trippel SB, Van Wyk JJ, Mankin HJ: Localization of somatomedin-C binding to bovine growth-plate chondrocytes in situ. J Bone Joint Surg Am 68:897902, 1986 28. Wise DR, Jennings AR: The development and morphology of the growth plates of two long bones of the turkey. Res Vet Sci 14:161166, 1973 29. West PG, Rowland GR, Budsberg SC, et al: Histomorphometric and angiographic analysis of bone healing the humerus of pigeons. Am J Vet Res 57:10101015, 1996 Address reprints requests to Thomas N. Tully, Jr., DVM, MS, Dipl ABVP (Avian) LSU-School of Veterinary Medicine Department of Veterinary Clinical Sciences Skip Bertman Drive Baton Rouge, LA 70803 e-mail: drtbird@aol.com