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The healing potential of the periosteum
Injury, Int. J. Care Injured (2005) 36S, S13—S19
www.elsevier.com/locate/injury
The healing potential of the periosteum Molecular aspects Konstantinos N. Malizos *, Loukia K. Papatheodorou Orthopaedic Department, University Hospital of Larissa, P.O. Box 1425, 41110 Larissa, Greece Accepted 25 July 2005
KEYWORDS Fracture; Periostium; Regeneration; Growth factors
Summary The presence of pluripotential mesenchymal cells in the under surface of the periosteum in combination with growth factors regularly produced or released after injury, provide this unique tissue with an important role in the healing of bone and cartilage. The periosteum contributes in the secondary callus formation with cells and growth factors and should always be preserved and protected when surgery is performed for the management of a fracture. The current evidence about the cellular interactions, the stimulants and the signalling pathways related to osteogenesis and chondrogenesis is described. An essential knowledge of the basics related to the contribution of the periosteum in the healing of fractures, osteotomies, during the process of distraction osteogenesis and in some degree in the repair of cartilagenous defects, provides the surgeons with a better insight to understand the upcoming ‘‘biological’’ interventions in the management of skeletal injuries. # 2005 Elsevier Ltd. All rights reserved.
Introduction Periosteum is a specialised connective tissue forming a thin but tough fibrous membrane firmly anchored to bone. The structure of periosteum varies with age. In infants and children is thicker, more vascular, active and loosely attached. In adults is thinner, inactive and more firmly adherent. The periosteum is a well vascularised ‘‘osteogenic organ’’.38 In adults the bone-forming potential of the periosteum is reactivated by trauma, infection and also in some cases of growing * Corresponding author. Tel.: +30 2410 682722/30 2410 681199; fax: +30 2410 670107. E-mail addresses: [email protected], [email protected], [email protected] (K.N. Malizos).
tumors. Many studies have shown that periosteum regenerates both cartilage and bone from its progenitor cells. The purpose of this article is to provide a brief overview of the healing potential of the periosteum.
Anatomy and histological considerations Periosteum is a specialised connective tissue anchored to bone through Sharpey’s fibers. These fibers represent a direct continuation of the periosteal collagen fibers around which the cortical lamellae have grown. The structure of periosteum varies with age.
0020–1383/$ — see front matter # 2005 Elsevier Ltd. All rights reserved. doi:10.1016/j.injury.2005.07.030
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The periosteum consists of two discrete layers: the outer fibrous layer containing fibroblasts, vessels and fibers of Sharpey and the inner cambium layer containing nerves, capillaries, osteoblasts and undifferentiated mesenchymal stem cells (MSCs). It is a well vascularised osteogenic organ with its blood supply deriving from arterioles and capillaries piercing the cortex to enter the medullary canal. The periosteal vessels are nourishing the outer one third of the diaphysis and complements the epiphysio-metaphyseal vessels and the principal nutrient arteries in the medullary cavity.12,24 The cambium layer serves as a reservoir of undifferentiated pluripotential mesenchymal cells, able to differentiate into chondrogenic and osteoblastic lineages and as a source of growth factors playing important role in the healing and remodelling process at the outer surface of the cortical bone.24,29,43,48,51,60,67 During embryogenesis these mesenchymal stem cells differentiate into neochondrocytes to produce cartilage tissue that is later replaced by bone. In children the cambium layer contributes to increasing the diameter of the bone during the growth period with differentiation of the mesenchymal stem cells in the periosteum directly to osteoblasts.60,62 In adults the bone-forming potential of the periosteum is reactivated by trauma, infection and also in some cases of growing tumors. Many studies have shown that periosteum regenerates both cartilage and bone from its progenitor cells.12,49,51,52,58,67,71,74
The role of periosteum in bone healing ‘‘Callus’’ formation after a fracture or an osteotomy is the result of a coordinated proliferation of various cell lineages of inflammatory cells, angioblasts, fibroblasts, chondroblasts and osteoblasts.41,63 Immediately following a bone fracture the vascular disruption is creating a haematoma into which a sequence of biochemical and cellular events commence to induce an inflammatory response. Activated platelet aggregates creating a barrier to limit blood loss at the site of injury. During the formation of the haematoma, platelet degranulation also takes place and a myriad of factors are released playing a crucial role in the healing process. These factors are chemo-attractant for the mesenchymal cells of the external soft tissue and the bone marrow.4,76 The highly abundant growth factors deriving from platelets can stimulate the proliferation and differentiation of the MSCs of periosteum.
K.N. Malizos, L.K. Papatheodorou
They also interact with the endothelial cells, attract granulocytes and macrophages to the fracture haematoma thus modulating the inflammatory reaction, which represents a crucial step of the healing process.4,25
Thrombin and platelet-derived factors The initial cellular event in the fracture healing process is the proliferation of periosteal and mesenchymal cells in the fracture haematoma and the soft tissues surrounding the fracture. Platelet-derived growth factor (PDGF) and basic fibroblast growth factor (bFGF) from the disrupted platelets enhance the mitogenic activity of fibroblasts39, osteoblasts10, smooth muscle cells31 and endothelial cells39 stimulate angiogenesis and further aggregation of platelets.35,36 They can also stimulate the proliferation of periosteum-derived cells and may contribute to the mitogenic response of the periosteum in the early stages of bone repair.20,21,64 After proliferation they differentiate to osteoblasts or chondroblasts to form bone and cartilage tissue prior to bridging of the fracture gap. The cartilaginous callus is gradually replaced into bone through endochondral ossification.
Cytokines The fracture-soft tissue haematoma around the site of the injury appears to be a potentially important source of immuno-modulatory cytokines. The regulation of various cell populations orchestrating the process of fracture healing depends on the biological effect evoked by the locally released cytokines and growth factors. Fractures create an inflammatory local environment with the early cell-associated mediators such as interleukin-1 (IL-1) to be confined to wound, where the abundant multiple soluble cytokines such as IL-6, IL-8, and IL-11 to probably be exported, thus exerting a combined impact on the monocytes of the peripheral blood where they initiate a complex response. At the early stages, macrophages release IL-1b and tumor necrosis factor-a (TNFa), which stimulate the resorption of the edges of necrotic bone by stimulating hematopoietic stem cells to differentiate into osteoclasts. At the later stages IL-1b and TNF-a regulate the number of osteoblasts. The role of programmed cell death (apoptosis) of osteoblasts as a normal concomitant of bone healing has been confirmed. Apoptotic cells markedly increase in the stage of intra-membranous bone formation and in chondrogenesis. Evidence was
The healing potential of the periosteum
found suggesting that IL-1b mediated the appearance and disappearance of osteoblasts, possibly by affecting the rates of differentiation and apoptosis, respectively. It has been proven that IL-1b and TNFa regulate the number of osteoblasts by up-regulating the Fas-mediated apoptosis of osteoblasts.68 Similarly chondrocytes involved in endochondral ossification undergo apoptosis. These early events in fracture healing may be initiated by the expression of early response genes such as c-fos.11 Understanding these mechanisms, conceivably could lead to the ability to control osteoblast levels at an injury site.47
Growth factors In the initiation of the bone repair process a crucial factor is the restoration of the blood flow to the fracture site. A potent angiogenic stimulator is the vascular endothelial growth factor (VEGF) that plays an important role in these processes.14,19 In the early stages of bone repair large amounts of VEGF are found in the fracture haematoma, a VEGF source that is not present in developing bones. This is highlighting the differences in endochondral ossification during development versus that in fracture repair.66 It is secreted by the endothelial and osteoblastic cells and besides angiogenesis is also maintaining the osteoblast function. It has been demonstrated that osteoblastic cells have receptors for VEGF.8,9 Therefore, this is suggestive that VEGF isoforms not only mediate bone vascularisation but also affect differentiation of progenitor cells to osteoblasts and hypertrophic chondrocytes.33 It has also been proven that VEGF is involved in the conversion of the soft cartilagenous callus to bony callus during fracture repair, just as VEGF couples angiogenesis, cartilage resorption and ossification in the growth plate of developing mice.18,66 Recently it was demonstrated the importance of VEGF-A isoform on bone formation and fracture healing acting in synergy with the BMP-4 and affecting different steps of cartilage and bone formation, suggesting multiple action mechanisms of VEGF.54 In vitro analysis of transgene effects on cellular behaviour, illustrated that VEGF-A acts as an autocrine factor for osteoblast differentiation but can also promote sprouting of endothelial cells, demonstrating a paracrine role in blood vessel formation.37 Bone morphogenetic proteins (BMPs) may be the main signal that regulates the skeletal repair. They act on progenitor cells to induce differentiation into osteoblasts and chondroblasts.6,13,57,61,69,73 The investigation on the osteogenic activity of the different types of BMPs in osteoblastic progenitor cells
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suggest that BMP-2, 4, 6, 7 and 9 may play an important role in inducing osteoblast differentiation of the mesenchymal stem cells. In contrast, most BMPs are able to stimulate osteogenesis in mature osteoblasts. The effect of rhBMP-2, rxBMP-4, and rhPDGF-bb as chemoattractive proteins for primary human mesenchymal progenitor cells (MPC), including the change in response to growth factors after differentiation, suggests a functional role of BMPs for recruitment of MPCs during bone development and remodelling, as well as in fracture healing.26,30 Osteocytes possibly participate in the initiation of chondrogenesis/osteogenesis and also have potential for the trans-differentiation of periosteal osteoblasts.29 Osteocytes may secret sclerostin which is a protein competing with type I and type II BMP receptors for binding to BMPs, thus disrupting BMP signalling towards periosteal cells to inhibit chondrogenic differentiation. In contrast, the periosteum underlaid by the cortical bone with dead osteocytes may differentiate into a chondrogenic lineage because of the lack of any competition of BMP signalling.72 This is explaining the radiological finding of the ‘‘periosteal bone line’’ appearing in case of a haematogenous osteomyelitis or other bone pathology, where the obliteration of the microcirculation into the haversian system induces septic necrosis of the affected bone cortex and elevation of the periosteum. Due to the death of the osteocytes in the underlying cortex and the lack of sclerostin, the BMPs bind to their receptors on the periosteal cells and initiate signalling for proliferation and differentiation to chondrogenic and subsequently to osteogenic lineages.
The role of prostaglandins The regulation of mesenchymal cell differentiation in bone repair has been investigated by Zhang X et al.75 who proposed a cyclo-oxygenase-2 (COX-2) mediated mechanism. According to his model, under basal conditions COX-2 activity maintains a population of mesenchymal cells in a pre-osteoblast state responsive to other osteoblastic signals. Immediately after injury, COX-2 formation is induced at the fracture site in the early phase of the reparative process of the bone and produces increased amounts of PGE2 in the local milieu. The PGE2 has been demonstrated to induce BMP-7 in the osteoblast cell line U2-OS. In other experiments it was found that PGE2 and BMP2 have an additive effect on osteoblastogenesis, providing evidence of an independent role of these factors. COX-2 regulates Runx2 (cbfa1) and Osx (osterix) expression during osteoblastogenesis, two essential transcription
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factors that are involved in regulating the multistep molecular pathway of osteoblast differentiation for both endochondral and intramembranous bone formation.44 They are expressed selectively and at high levels in osteoblasts and also in the periosteum and the perichondrium.7 Runx2 and its companion subunit Cbfb are needed for an early step in this pathway, whereas Osx is required for the differentiation of preosteoblasts to fully functioning osteoblasts.44 Furthermore, Osx (osterix) is highly specific to osteoblast in vivo and acts downstream of Runx2 to induce osteoblastic differentiation in bipotential chondro- & osteo-progenitor cells.45
Clinical and experimental applications In clinical practice apart from the fractures and the osteotomies where the healing process follows the steps described above, neo-osteogenesis is taking place in the process known as ‘‘distraction osteogenesis’’, where mechanical elongation is applied at a constant rate for a definite period of time. Three different modes of ossification are developing in parallel during the phase of distraction and the phase of maturation: the intamembranous, the endochondral and the transchondral (a mixed mode of transmembranous and endochondral).29 The stimulatory effect of low intensity ultrasound on bone healing is known since the middle of the 20th century. In a recent in vitro study, Low Intensity Pulsed Ultrasound (LipUs) was applied directly in human periosteal cell line and induced proliferation, differentiation and osteogenesis. There were also more calcified nodules produced into
K.N. Malizos, L.K. Papatheodorou
the culture media. The authors postulated that the LipUs modulate the differentiated cells by the micromechanical stimulation loop that simulates the bone remodelling mechanism.28 In an other experimental application, where the LipUs transducers had been directly attached on the periosteum of osteotomised sheep tibiae, it enhanced periosteal healing and accelerated the development of strength and stiffness of the callus.22,55 The osteogenic potential of the periosteum has been investigated also in heterotopic conditions, but with the preservation of its blood supply. Periosteum elevated as vascularised periosteal flap in an experimental study in rabbits, demonstrated strong osteogenic capacity. Combined with bone grafts resulted in larger specimens. This can be a ‘‘living tissue engineering’’ alternative for the reconstruction of small skeletal defects and defects with a poorly vascularised tissue environment.7 In another application in humans, the elevated and ‘‘reversed like a sleeve’’ periosteum at the implanted end of a vascularised fibular graft, was examined on specimens retrieved from femoral heads with osteonecrosis, when they were converted to total hip arthroplasty. The obtained histological sections after double tetracycline labelling demonstrated an increased osteogenic potential of the reversed segment of the periosteum (Fig. 1A—C).34
The role of periosteum in chondrogenesis In vitro and in vivo studies have shown that cartilage formation is taking place in the cambium layer of
Figure 1 The osteogenic potential of the activated periosteum in a tissue enviroment with mesenchymal progenitor cells and blood supply. (A) Osteonecrosis of femoral head treated with implantation of a vascularised fibula. (B) The periosteum of the fibula was reversed (green arrows) at implantation so that the cambium layer was exposed to direct contact with the cancellous graft. (C) A longitudinal histologic section processed without decalcification after double tetracycline labelling. The excessive neo—osteogenesis on the cambium layer of the reversed periosteum is demonstrated (white arrows).
The healing potential of the periosteum
the periosteum and the chondrocyte precursor cells also reside in this location.12,42,51,52,58,70,71,74 Chondrogenesis commences in the juxtaosseous area of the cambium layer and progresses to the juxtafibrous region. Neocartilage growth is appositional, displacing the fibrous layer away from the cartilage already formed, as new cartilage is formed between these two layers.24 The development and maturation of neochondrocytes occur in three sequential phases distinguished by transition-restriction points and regulated separately by growth factors synthesised by the periosteum in conditions conducive to chondrogenesis and possibly with mechanical stimulation or other factors.48 These include transforming growth factor-beta 1 (TGFb-1), insulin like growth factor-1 (IGF-1), growth and differentiation factor-5 (GDf-5), bone morphogenetic protein-2 (BMP-2), integrins, and the receptors for these molecules.15,41,48,65 The IGF-1 and TGFb-1 regulate proliferation and differentiation of periosteal mesenchymal cells during chondrogenesis.16 IGF-1 can induce early differentiation and maintain proliferation and phenotype. The TGFb-1 induces early differentiation and proliferation toward chondrogenesis but may not have a potent effect on proliferation and type II collagen expression in the later stages of periosteal chondrogenesis.41 Furthermore, the addition of mitogen fibroblast growth factor-2 (FGF-2) during early stage of the in vitro culture of periosteum in the presence of TGFb-1 enhances cell proliferation, which results in increased neo-cartilage formation at later stages.40,65 The basic fibroblast growth factor (bFGF) stimulates proliferation of chondrocytes but may inhibit chondrocyte terminal differentiation. Periosteum can serve as a potential matrix for use in general tissue engineering applications. Because it produces chondrogenic bioactive factors, it can be transplanted as a whole tissue and it can serve as its own scaffold or matrix onto which other cells or growth factors, or both can be adhered. Cartilage repair is possible with transplantation of periosteal grafts. Periosteum has been used alone for biologic resurfacing arthroplasty in humans for more than a decade and in combination with continuous passive motion (CPM) to stimulate joint regeneration neochondrogenesis.23,27,46,50,59 As cell density varies at different sites in the periosteum, its chondrogenic potential varies with the donor sites in proportion to the total number of stem cells in the cambium layer. With aging, the chondrogenic potential of periosteum decreases significantly as the number of chondrocyte precursors declines in the cambium layer.17,53
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In a recent study, it was found that although the articular chondrocytes are able to form clones of different properties (in agarose), the periosteum has a capacity of stimulating clonal growth and differentiation of the chondrocytes and secretes significant amounts of IL-6, IL-8, GM-CSF and TGFb. This supports the view that the repair of cartilage defects with seeded chondrocytes could benefit from the combination with a periosteal graft, as the production of TGFb from the implanted chondrocytes could induce periosteal chondrogenesis and together with the matrix from implanted chondrocyte production a repair of cartilagenous appearance may develop as a result of dual chondrogenic response.5 In a number of observational studies periosteal grafts have been utilised to repair defects of the cartilage on the surface of the patella and the femoral condyles with satisfactory outcome for the first 3—4 years in 53—70% of the patients. In the immediate postoperative period the application of continuous passive motion may enhance the formation of ‘‘hyaline-like’’ cartilage.1—3,23,27,32,56
Future applications The histological characteristics of the periosteum makes it a unique tissue with a unique capacity to be engineered. It could be both, a natural scaffold and/ or a source of cells and bioactive factors, which might serve as a transplantable unit with negligible donor site morbidity. Elevating vascularised periosteal flaps from the surface of large bones we could obtain useful living scaffolds for auto-transplantation with enhanced healing potential. The availability of recombinant growth factors that could modulate the healing process according to the needs for reconstruction is a challenge for further research.
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44. Nakashima K, Crombrugghe B. Transcriptional mechanisms in osteoblast differentiation and bone formation. Trends Genet 2003;19(8):458—66. 45. Nakashima K, Zhou X, Kunkel G, et al. The novel zing fingercontaining transcription factor osterix is required for osteoblast differentiation and bone formation. Cell 2002;108: 17—29. 46. Niedermann B, Boe S, Lauritzen J, Rubak JM. Glued periosteal grafts in the knee. Acta Orthop Scand 1985;56:457—60. 47. Olmedo ML, Landry PS, Sadasivan KK, et al. Regulation of osteoblast levels during bone healing. J Orthop Trauma 1999;13(5):356—62. 48. O’Driscoll SW, Fitzsimmons JS. The role of periosteum in cartilage repair. Clin Orthop 2001;(Suppl. 391):S190—207. 49. O’Driscoll SW, Keeley FW, Salter RB. The chondrogenic potential of free autogenous periosteal grafts for biological resurfacing of major full-thickness defects in joint surfaces under the influence of continuous passive motion. J Bone Joint Surg 1986;68A:1017—35. 50. O’Driscoll SW, Keeley FW, Salter RB. Durability of regenerated articular cartilage produced by free autogenous periosteal grafts in major full-thickness defects in joint surfaces under the influence of continuous passive motion. A followup report at one year. J Bone Joint Surg Am 1988;70(4):595— 606. 51. O’Driscoll SW, Recklies Ad. Poole AR. Chondrogenesis in periosteal explants: an organ culture model for in vitro study. J Bone Joint Surg 1994;76A:1042—51. 52. O’Driscoll SW, Salter RB. The induction of neochondrogenesis in free intra-articular periosteal autografts under the influence of continuous passive motion: an experimental investigation in the rabbit. J Bone Joint Surg 1984;66A:1248—57. 53. O’Driscoll SW, Saris DB, Ito Y, Fitzimmons JS. The chondrogenic potential of periosteum decreases with age. J Orthop Res 2001;19(1):95—103. 54. Peng H, Wright V, Usas A, et al. Synergistic enhancement of bone formation and healing by stem cell-expressed VEGF and bone morphogenetic protein-4. J Clin Invest 2002;110:751—9. 55. Protoppapas VC, Baga D, Fotiadis DI, et al. An ultrasound wearable system for the monitoring and acceleration of fracture healing in long bones. IEEE Trans Biomed Eng 2005;52:1597—608. 56. Ritsila VA, Santavirta S, Alhopuro S, et al. Periosteal and perichondral grafting in reconstructive surgery. Clin Orthop 1994;302:259—65. 57. Rosen V, Cox K, Hattersley G. Bone morphogenetic protein. In: Bilezikian JP, Raisz LG, Rodan GA., editors. Principles of bone biology. San Diego, CA: Academic Press; 1996. p. 661— 71. 58. Rubak JM. Osteochondrogenesis of free periosteal grafts in the rabbit iliac crest. Acta Orthop Scand 1983;54:826—31. 59. Salter RB. The biologic concept of continuous passive motion of synovial joints. The first 18 years of basic research and its clinical application. Clin Orthop Relat Res 1989;242:12—25.
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60. Sandberg M, Aro H, Multimaki P, Aho H, Vuorio E. In situ localization of collagen production by chondrocytes and osteoblasts in fracture callus. J Bone Joint Surg 1989;71: 69—77. 61. Schmitt JM, Hwang K, Winn SR, Hollinger JO. Bone morphogenetic proteins: an update on basic biology and clinical relevance. J Orthop Res 1999;17:269—78. 62. Scott-Savage P, Hall BK. Differentiative ability of the tibial periosteum for the embryonic chick. Acta Anat (Basel) 1980;106:129—40. 63. Simmons DJ. Fracture healing perspectives. Clin Orthop 1985;200:100—13. 64. Slater M, Patava J, Kingham K, Mason RS. Involvement of platelets in stimulating osteogenic activity. J Orthop Res 1995;13(5):655—63. 65. Stevens MM, Marini RP, Martin I, et al. FGF-2 enhances TGF-beta1-induced periosteal chondrogenesis. J Orthop Res 2004;22(5):1114—9. 66. Street J, Bao M, deGuzman L, et al. Vascular endothelial growth factor stimulates bone repair by promoting angiogenesis and bone turnover. Proc Nat Acad Sci USA 2002;23; 99(15):9656—9661. 67. Thorogood P. In vitro studies on skeletogenic potential of membrane bone periosteal cells. J Embryol Exp Morphol 1979;54:185—207. 68. Tsuboi M, Kawakami A, Nakashima T, et al. Tumor necrosis factor-alpha and interleukin-1beta increase the Fasmediated apoptosis of human osteoblasts. J Lab Clin Med 1999;134(3):222—31. 69. Urist MR. Bone: formation by autoinduction. Science 1965;150:893—9. 70. Vachon A, McIlwraith CW, Trotter GW, et al. Neochondrogenesis in free intra-articular, periosteal, and perichondrial autografts in horses. Am J Vet Res 1989;50:1787—94. 71. Wakitani S, Goto T, Pineda SJ, et al. Mesenchymal cell-based repair of large, full-thickness defects of articular cartilage. J Bone Joint Surg 1994;76A:579—92. 72. Winkler DG, Sutherland MK, Geoghegan JC, et al. Osteocyte control of bone formation via sclerostin, a novel BMP antagonist. EMBO J 2003;22:6267—76. 73. Wozney JM, Rosen V, Celeste A, et al. Novel regulators of bone formation: molecular clones and activities. Science 1988;242:1528—34. 74. Zarnett R, Delaney JP, Driscoll SW, Salter RB. The cellular origin of neochodrogenesis in major full-thickness defects of a joint surface treated by autogenous periosteal grafts and subjected to continuous passive motion. Clin Orthop 1987;222:267—74. 75. Zhang X, Schwarz EM, Young DA, et al. Cyclooxygenase-2 regulates mesenchymal cell differentiation into the osteoblast lineage and is critically involved in bone repair. J Clin Invest 2002;109(11):1405—15. 76. Zuo ZJ, Zhu JZ. Study on the microstructures of skull fracture. Forensic Sci Int 1991;50(1):1—14.
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The healing potential of the periosteum Molecular aspects Konstantinos N. Malizos *, Loukia K. Papatheodorou Orthopaedic Department, University Hospital of Larissa, P.O. Box 1425, 41110 Larissa, Greece Accepted 25 July 2005
KEYWORDS Fracture; Periostium; Regeneration; Growth factors
Summary The presence of pluripotential mesenchymal cells in the under surface of the periosteum in combination with growth factors regularly produced or released after injury, provide this unique tissue with an important role in the healing of bone and cartilage. The periosteum contributes in the secondary callus formation with cells and growth factors and should always be preserved and protected when surgery is performed for the management of a fracture. The current evidence about the cellular interactions, the stimulants and the signalling pathways related to osteogenesis and chondrogenesis is described. An essential knowledge of the basics related to the contribution of the periosteum in the healing of fractures, osteotomies, during the process of distraction osteogenesis and in some degree in the repair of cartilagenous defects, provides the surgeons with a better insight to understand the upcoming ‘‘biological’’ interventions in the management of skeletal injuries. # 2005 Elsevier Ltd. All rights reserved.
Introduction Periosteum is a specialised connective tissue forming a thin but tough fibrous membrane firmly anchored to bone. The structure of periosteum varies with age. In infants and children is thicker, more vascular, active and loosely attached. In adults is thinner, inactive and more firmly adherent. The periosteum is a well vascularised ‘‘osteogenic organ’’.38 In adults the bone-forming potential of the periosteum is reactivated by trauma, infection and also in some cases of growing * Corresponding author. Tel.: +30 2410 682722/30 2410 681199; fax: +30 2410 670107. E-mail addresses: [email protected], [email protected], [email protected] (K.N. Malizos).
tumors. Many studies have shown that periosteum regenerates both cartilage and bone from its progenitor cells. The purpose of this article is to provide a brief overview of the healing potential of the periosteum.
Anatomy and histological considerations Periosteum is a specialised connective tissue anchored to bone through Sharpey’s fibers. These fibers represent a direct continuation of the periosteal collagen fibers around which the cortical lamellae have grown. The structure of periosteum varies with age.
0020–1383/$ — see front matter # 2005 Elsevier Ltd. All rights reserved. doi:10.1016/j.injury.2005.07.030
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The periosteum consists of two discrete layers: the outer fibrous layer containing fibroblasts, vessels and fibers of Sharpey and the inner cambium layer containing nerves, capillaries, osteoblasts and undifferentiated mesenchymal stem cells (MSCs). It is a well vascularised osteogenic organ with its blood supply deriving from arterioles and capillaries piercing the cortex to enter the medullary canal. The periosteal vessels are nourishing the outer one third of the diaphysis and complements the epiphysio-metaphyseal vessels and the principal nutrient arteries in the medullary cavity.12,24 The cambium layer serves as a reservoir of undifferentiated pluripotential mesenchymal cells, able to differentiate into chondrogenic and osteoblastic lineages and as a source of growth factors playing important role in the healing and remodelling process at the outer surface of the cortical bone.24,29,43,48,51,60,67 During embryogenesis these mesenchymal stem cells differentiate into neochondrocytes to produce cartilage tissue that is later replaced by bone. In children the cambium layer contributes to increasing the diameter of the bone during the growth period with differentiation of the mesenchymal stem cells in the periosteum directly to osteoblasts.60,62 In adults the bone-forming potential of the periosteum is reactivated by trauma, infection and also in some cases of growing tumors. Many studies have shown that periosteum regenerates both cartilage and bone from its progenitor cells.12,49,51,52,58,67,71,74
The role of periosteum in bone healing ‘‘Callus’’ formation after a fracture or an osteotomy is the result of a coordinated proliferation of various cell lineages of inflammatory cells, angioblasts, fibroblasts, chondroblasts and osteoblasts.41,63 Immediately following a bone fracture the vascular disruption is creating a haematoma into which a sequence of biochemical and cellular events commence to induce an inflammatory response. Activated platelet aggregates creating a barrier to limit blood loss at the site of injury. During the formation of the haematoma, platelet degranulation also takes place and a myriad of factors are released playing a crucial role in the healing process. These factors are chemo-attractant for the mesenchymal cells of the external soft tissue and the bone marrow.4,76 The highly abundant growth factors deriving from platelets can stimulate the proliferation and differentiation of the MSCs of periosteum.
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They also interact with the endothelial cells, attract granulocytes and macrophages to the fracture haematoma thus modulating the inflammatory reaction, which represents a crucial step of the healing process.4,25
Thrombin and platelet-derived factors The initial cellular event in the fracture healing process is the proliferation of periosteal and mesenchymal cells in the fracture haematoma and the soft tissues surrounding the fracture. Platelet-derived growth factor (PDGF) and basic fibroblast growth factor (bFGF) from the disrupted platelets enhance the mitogenic activity of fibroblasts39, osteoblasts10, smooth muscle cells31 and endothelial cells39 stimulate angiogenesis and further aggregation of platelets.35,36 They can also stimulate the proliferation of periosteum-derived cells and may contribute to the mitogenic response of the periosteum in the early stages of bone repair.20,21,64 After proliferation they differentiate to osteoblasts or chondroblasts to form bone and cartilage tissue prior to bridging of the fracture gap. The cartilaginous callus is gradually replaced into bone through endochondral ossification.
Cytokines The fracture-soft tissue haematoma around the site of the injury appears to be a potentially important source of immuno-modulatory cytokines. The regulation of various cell populations orchestrating the process of fracture healing depends on the biological effect evoked by the locally released cytokines and growth factors. Fractures create an inflammatory local environment with the early cell-associated mediators such as interleukin-1 (IL-1) to be confined to wound, where the abundant multiple soluble cytokines such as IL-6, IL-8, and IL-11 to probably be exported, thus exerting a combined impact on the monocytes of the peripheral blood where they initiate a complex response. At the early stages, macrophages release IL-1b and tumor necrosis factor-a (TNFa), which stimulate the resorption of the edges of necrotic bone by stimulating hematopoietic stem cells to differentiate into osteoclasts. At the later stages IL-1b and TNF-a regulate the number of osteoblasts. The role of programmed cell death (apoptosis) of osteoblasts as a normal concomitant of bone healing has been confirmed. Apoptotic cells markedly increase in the stage of intra-membranous bone formation and in chondrogenesis. Evidence was
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found suggesting that IL-1b mediated the appearance and disappearance of osteoblasts, possibly by affecting the rates of differentiation and apoptosis, respectively. It has been proven that IL-1b and TNFa regulate the number of osteoblasts by up-regulating the Fas-mediated apoptosis of osteoblasts.68 Similarly chondrocytes involved in endochondral ossification undergo apoptosis. These early events in fracture healing may be initiated by the expression of early response genes such as c-fos.11 Understanding these mechanisms, conceivably could lead to the ability to control osteoblast levels at an injury site.47
Growth factors In the initiation of the bone repair process a crucial factor is the restoration of the blood flow to the fracture site. A potent angiogenic stimulator is the vascular endothelial growth factor (VEGF) that plays an important role in these processes.14,19 In the early stages of bone repair large amounts of VEGF are found in the fracture haematoma, a VEGF source that is not present in developing bones. This is highlighting the differences in endochondral ossification during development versus that in fracture repair.66 It is secreted by the endothelial and osteoblastic cells and besides angiogenesis is also maintaining the osteoblast function. It has been demonstrated that osteoblastic cells have receptors for VEGF.8,9 Therefore, this is suggestive that VEGF isoforms not only mediate bone vascularisation but also affect differentiation of progenitor cells to osteoblasts and hypertrophic chondrocytes.33 It has also been proven that VEGF is involved in the conversion of the soft cartilagenous callus to bony callus during fracture repair, just as VEGF couples angiogenesis, cartilage resorption and ossification in the growth plate of developing mice.18,66 Recently it was demonstrated the importance of VEGF-A isoform on bone formation and fracture healing acting in synergy with the BMP-4 and affecting different steps of cartilage and bone formation, suggesting multiple action mechanisms of VEGF.54 In vitro analysis of transgene effects on cellular behaviour, illustrated that VEGF-A acts as an autocrine factor for osteoblast differentiation but can also promote sprouting of endothelial cells, demonstrating a paracrine role in blood vessel formation.37 Bone morphogenetic proteins (BMPs) may be the main signal that regulates the skeletal repair. They act on progenitor cells to induce differentiation into osteoblasts and chondroblasts.6,13,57,61,69,73 The investigation on the osteogenic activity of the different types of BMPs in osteoblastic progenitor cells
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suggest that BMP-2, 4, 6, 7 and 9 may play an important role in inducing osteoblast differentiation of the mesenchymal stem cells. In contrast, most BMPs are able to stimulate osteogenesis in mature osteoblasts. The effect of rhBMP-2, rxBMP-4, and rhPDGF-bb as chemoattractive proteins for primary human mesenchymal progenitor cells (MPC), including the change in response to growth factors after differentiation, suggests a functional role of BMPs for recruitment of MPCs during bone development and remodelling, as well as in fracture healing.26,30 Osteocytes possibly participate in the initiation of chondrogenesis/osteogenesis and also have potential for the trans-differentiation of periosteal osteoblasts.29 Osteocytes may secret sclerostin which is a protein competing with type I and type II BMP receptors for binding to BMPs, thus disrupting BMP signalling towards periosteal cells to inhibit chondrogenic differentiation. In contrast, the periosteum underlaid by the cortical bone with dead osteocytes may differentiate into a chondrogenic lineage because of the lack of any competition of BMP signalling.72 This is explaining the radiological finding of the ‘‘periosteal bone line’’ appearing in case of a haematogenous osteomyelitis or other bone pathology, where the obliteration of the microcirculation into the haversian system induces septic necrosis of the affected bone cortex and elevation of the periosteum. Due to the death of the osteocytes in the underlying cortex and the lack of sclerostin, the BMPs bind to their receptors on the periosteal cells and initiate signalling for proliferation and differentiation to chondrogenic and subsequently to osteogenic lineages.
The role of prostaglandins The regulation of mesenchymal cell differentiation in bone repair has been investigated by Zhang X et al.75 who proposed a cyclo-oxygenase-2 (COX-2) mediated mechanism. According to his model, under basal conditions COX-2 activity maintains a population of mesenchymal cells in a pre-osteoblast state responsive to other osteoblastic signals. Immediately after injury, COX-2 formation is induced at the fracture site in the early phase of the reparative process of the bone and produces increased amounts of PGE2 in the local milieu. The PGE2 has been demonstrated to induce BMP-7 in the osteoblast cell line U2-OS. In other experiments it was found that PGE2 and BMP2 have an additive effect on osteoblastogenesis, providing evidence of an independent role of these factors. COX-2 regulates Runx2 (cbfa1) and Osx (osterix) expression during osteoblastogenesis, two essential transcription
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factors that are involved in regulating the multistep molecular pathway of osteoblast differentiation for both endochondral and intramembranous bone formation.44 They are expressed selectively and at high levels in osteoblasts and also in the periosteum and the perichondrium.7 Runx2 and its companion subunit Cbfb are needed for an early step in this pathway, whereas Osx is required for the differentiation of preosteoblasts to fully functioning osteoblasts.44 Furthermore, Osx (osterix) is highly specific to osteoblast in vivo and acts downstream of Runx2 to induce osteoblastic differentiation in bipotential chondro- & osteo-progenitor cells.45
Clinical and experimental applications In clinical practice apart from the fractures and the osteotomies where the healing process follows the steps described above, neo-osteogenesis is taking place in the process known as ‘‘distraction osteogenesis’’, where mechanical elongation is applied at a constant rate for a definite period of time. Three different modes of ossification are developing in parallel during the phase of distraction and the phase of maturation: the intamembranous, the endochondral and the transchondral (a mixed mode of transmembranous and endochondral).29 The stimulatory effect of low intensity ultrasound on bone healing is known since the middle of the 20th century. In a recent in vitro study, Low Intensity Pulsed Ultrasound (LipUs) was applied directly in human periosteal cell line and induced proliferation, differentiation and osteogenesis. There were also more calcified nodules produced into
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the culture media. The authors postulated that the LipUs modulate the differentiated cells by the micromechanical stimulation loop that simulates the bone remodelling mechanism.28 In an other experimental application, where the LipUs transducers had been directly attached on the periosteum of osteotomised sheep tibiae, it enhanced periosteal healing and accelerated the development of strength and stiffness of the callus.22,55 The osteogenic potential of the periosteum has been investigated also in heterotopic conditions, but with the preservation of its blood supply. Periosteum elevated as vascularised periosteal flap in an experimental study in rabbits, demonstrated strong osteogenic capacity. Combined with bone grafts resulted in larger specimens. This can be a ‘‘living tissue engineering’’ alternative for the reconstruction of small skeletal defects and defects with a poorly vascularised tissue environment.7 In another application in humans, the elevated and ‘‘reversed like a sleeve’’ periosteum at the implanted end of a vascularised fibular graft, was examined on specimens retrieved from femoral heads with osteonecrosis, when they were converted to total hip arthroplasty. The obtained histological sections after double tetracycline labelling demonstrated an increased osteogenic potential of the reversed segment of the periosteum (Fig. 1A—C).34
The role of periosteum in chondrogenesis In vitro and in vivo studies have shown that cartilage formation is taking place in the cambium layer of
Figure 1 The osteogenic potential of the activated periosteum in a tissue enviroment with mesenchymal progenitor cells and blood supply. (A) Osteonecrosis of femoral head treated with implantation of a vascularised fibula. (B) The periosteum of the fibula was reversed (green arrows) at implantation so that the cambium layer was exposed to direct contact with the cancellous graft. (C) A longitudinal histologic section processed without decalcification after double tetracycline labelling. The excessive neo—osteogenesis on the cambium layer of the reversed periosteum is demonstrated (white arrows).
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the periosteum and the chondrocyte precursor cells also reside in this location.12,42,51,52,58,70,71,74 Chondrogenesis commences in the juxtaosseous area of the cambium layer and progresses to the juxtafibrous region. Neocartilage growth is appositional, displacing the fibrous layer away from the cartilage already formed, as new cartilage is formed between these two layers.24 The development and maturation of neochondrocytes occur in three sequential phases distinguished by transition-restriction points and regulated separately by growth factors synthesised by the periosteum in conditions conducive to chondrogenesis and possibly with mechanical stimulation or other factors.48 These include transforming growth factor-beta 1 (TGFb-1), insulin like growth factor-1 (IGF-1), growth and differentiation factor-5 (GDf-5), bone morphogenetic protein-2 (BMP-2), integrins, and the receptors for these molecules.15,41,48,65 The IGF-1 and TGFb-1 regulate proliferation and differentiation of periosteal mesenchymal cells during chondrogenesis.16 IGF-1 can induce early differentiation and maintain proliferation and phenotype. The TGFb-1 induces early differentiation and proliferation toward chondrogenesis but may not have a potent effect on proliferation and type II collagen expression in the later stages of periosteal chondrogenesis.41 Furthermore, the addition of mitogen fibroblast growth factor-2 (FGF-2) during early stage of the in vitro culture of periosteum in the presence of TGFb-1 enhances cell proliferation, which results in increased neo-cartilage formation at later stages.40,65 The basic fibroblast growth factor (bFGF) stimulates proliferation of chondrocytes but may inhibit chondrocyte terminal differentiation. Periosteum can serve as a potential matrix for use in general tissue engineering applications. Because it produces chondrogenic bioactive factors, it can be transplanted as a whole tissue and it can serve as its own scaffold or matrix onto which other cells or growth factors, or both can be adhered. Cartilage repair is possible with transplantation of periosteal grafts. Periosteum has been used alone for biologic resurfacing arthroplasty in humans for more than a decade and in combination with continuous passive motion (CPM) to stimulate joint regeneration neochondrogenesis.23,27,46,50,59 As cell density varies at different sites in the periosteum, its chondrogenic potential varies with the donor sites in proportion to the total number of stem cells in the cambium layer. With aging, the chondrogenic potential of periosteum decreases significantly as the number of chondrocyte precursors declines in the cambium layer.17,53
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In a recent study, it was found that although the articular chondrocytes are able to form clones of different properties (in agarose), the periosteum has a capacity of stimulating clonal growth and differentiation of the chondrocytes and secretes significant amounts of IL-6, IL-8, GM-CSF and TGFb. This supports the view that the repair of cartilage defects with seeded chondrocytes could benefit from the combination with a periosteal graft, as the production of TGFb from the implanted chondrocytes could induce periosteal chondrogenesis and together with the matrix from implanted chondrocyte production a repair of cartilagenous appearance may develop as a result of dual chondrogenic response.5 In a number of observational studies periosteal grafts have been utilised to repair defects of the cartilage on the surface of the patella and the femoral condyles with satisfactory outcome for the first 3—4 years in 53—70% of the patients. In the immediate postoperative period the application of continuous passive motion may enhance the formation of ‘‘hyaline-like’’ cartilage.1—3,23,27,32,56
Future applications The histological characteristics of the periosteum makes it a unique tissue with a unique capacity to be engineered. It could be both, a natural scaffold and/ or a source of cells and bioactive factors, which might serve as a transplantable unit with negligible donor site morbidity. Elevating vascularised periosteal flaps from the surface of large bones we could obtain useful living scaffolds for auto-transplantation with enhanced healing potential. The availability of recombinant growth factors that could modulate the healing process according to the needs for reconstruction is a challenge for further research.
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