Bone Healing in Hand Transplantation

Transcription

1 Section 9-a Bone Healing in Hand Transplantation Markus Gabl, Sigurd Pechlaner, Martin Lutz, Rohit Arora, Michael Blauth, Michael Rieger, Marina Ninkovic, Hildegunde Piza, Stefan Schneeberger, Raimund Margreiter Introduction Biology of Bone Healing Various bone disorders can affect the ability of bone cells to structure organic and inorganic components. Avascularity can cause osteonecrosis, with death of haematopoietic cells, lipocytes and endothelial cells. Repair of osteonecrosis is the time needed for the process to replace necrotic bone. Callous fracture healing is a regenerative process consisting of three stages of inflammation: development of soft callus, of hard callus and remodelling [1, 2]. During inflammation, new blood vessels are induced, enhancing angiogenesis, which can be investigated by Doppler ultrasound. Following inflammation, fibrous and cartilaginous tissue known as soft callus develops, which can be observed by grey-scale ultrasound. In the hard callus stage, cartilaginous tissue converts to woven bone, which will finally be remodelled to lamellar bone. In primary bone healing under rigid plate fixation creeping substitution can be observed histologically after 4 weeks. Following the Haversian system, osteoclast activities are first necessary to enable cone formations and ingrowth of bridging osteoblasts. This remodelling takes time and weakens the bone for 1 2 years. In the remaining tiny gaps, blood vessels and osteoblasts grow in within the first 2 weeks, forming a lamellar bone that is osteoconductive and bridged at week 4. Healing of Bone Grafts Healing of nonvascularised autologous, cancellous and cortical bone shows inflammatory response with vascular ingrowth. With increase of fibrous granulation, in 2 weeks, repair of cancellous grafts differs as osteoblastic new bone is apposed onto necrotic trabeculae, correlating radiographically with an increase in radiodensity. At month 6, this graft is completely repaired, with the necrotic trabeculae resorbed by osteoclasts. The osteoinductive and osteoconductive graft is initially stronger due to apposition of new bone, but strength declines to normal when the necrotic bone is resorbed. Nonvascularized autologous cortical grafts are incorporated by creeping substitution at a lower rate due to the greater amount of osteonecrosis. In humans, graft healing is prolonged, with loss of 50% graft strength within the first 6 months, maintaining this strength for another 6 months. Radiographically, density is reduced due to bone porosity. Graft strength can be regained up to the second year. In humans, fatigue failures occur between month 6 and 18 [3]. The osteoconductive graft is not completely substituted but remains as a mixture of necrotic donor bone and new host bone. Healing of the osteoconductive graft depends on compression and oxygenation, which can be improved by vascularisation. Such vascularised autologous cortical grafts contain less necrotic bone and show the identical pattern of repair. Strength and stiff-

2 272 M. Gabl, S. Pechlaner, M. Lutz et al. ness, however, were found to be accelerated, making them superior to nonvascularised grafts [4]. Allogeneic Bone Grafting In nonvascularised allogeneic cancellous bone, incorporation lasts longer, with increase of vascular response and with the granulation tissue becoming loosely structured. This web is filled with inflammatory cells rather than with fibroblasts and blood vessels. Bone resorption and bone formation are delayed, and the graft may incorporate incompletely. Nonvascularised allogeneic cortical grafts are osteoconductive and show creeping substitution to be markedly prolonged. Allografts differ from autografts, as vascular penetration and bone formation are slower and resorptive activity is more extensive. Primary lymphocytes dominate, and fibrous tissue encapsulates the graft. The inflammation can either disappear or become chronic. The initially vascular network around the graft becomes occluded, leading to periosteal necrosis and thereby prohibiting appositional bone healing, with more necrotic bone existing than new bone to be formed. Immunological Response to Allogeneic Structured Bone Grafts There is much evidence that bone is immunogenetic. The marrow contained in bone, endosteal and periosteal cell-surface antigens as well as bone matrix have been suggested to be responsible for immunogenicity [5]. Cell-mediated immunity is considered to play a minor role in rejection of composite tissue allografts and of bone alone as compared with antibody-mediated response. There is some evidence that cytotoxic antibodies directed against bone allografts do, indeed, appear and may coincide with cellular immunity although they seem to not be directly involved in the rejection process. Bone healing after allotransplantation may proceed normally. Chronic repair is characterised by greater incidence of nonunion or delayed union, peripheral resorption or loss of graft size. In some cases, the graft can be resorbed completely [1]. Vascularized Allogeneic Cortical Grafts Under Immunosuppression In contrast to avascular allografts, primary vascularisation of limb-tissue allograft is reported to change the pattern of rejection into considerable humeral response early after transplantation [6]. The various components interact with the host immune system in a complex pattern, eliciting less immune response than an individual tissue allograft. Radiographs and histology can be indistinguishable from autograft healing as long as sufficient immunosuppressive drugs are taken. After withdrawal of the immunosuppression, both vascularised and nonvascularised allografts can be rejected quickly [7]. In experimental studies with vascularised bone marrow transplantation, stromal and marrow cells act early after transplantation, circulate to the lymphopoietic system of the recipient and are reported to generate tolerance in long-term survival [8]. Factors affecting chimerism in bone allotransplantation are still unclear. Allogeneic vascularised knee joints have been transplanted under immunosuppression with cyclosporine and azathioprine and corticosteroids with good early results [9]. Biomechanical Properties of Bone Grafts Incorporation of cancellous bone grafts with new bone formation upon necrotic trabeculae results in early graft strength. In cortical bone grafts, initial graft resorption causes graft porosity with reduced strength, which only slowly improves. It is suggested that human segmental cortical bone grafts loose almost half of their biomechanical strength within the first 6 months and remain weakened for another 6 months. This hypothesis is supported by the high number of graft failures between 6 and 8 months after transplantation. Creeping substitution is significantly prolonged in allografts, with fracture of

3 Bone Healing in Hand Transplantation 273 large-segment allografts occurring after 26 months [10]. Vascularised bone allografts under immunosuppression show superior biological and biomechanical behaviour with higher rates of bone integration [11, 12]. Case Presentation In the following, we report on our experience in bone healing of our first patient with double hand transplantation [13]. Patient The hands of a 47-year-old policeman were traumatised severely by the explosion of a bomb he was trying to deactivate. Both hands had to be amputated at the wrist. Soft tissue coverage of the stumps was poor. Tendons and muscles of both forearms were retracted. Double hand transplantation was performed in March 2000 [14]. For bone reconstruction, a proximally based flap of the interosseous membrane together with the periosteum was created at both recipient forearms proximal to the osteotomy site, which was located at the distal third of the forearm. Donor forearm bones, which had a diameter 2 mm greater than the recipient bones, were stabilized to the recipient bones with compression using 7- and 8-hole low-contact dynamic compression plates and 3.5-mm screws. No additional autologous bone grafts were used. The periosteal flap was positioned to cover the osteotomy sites before vascular reconstruction of the transplanted limb was completed. Forearms were splinted for 4 weeks to protect tendon healing. Induction therapy with antithymocyte globulin (Fresenius Medical Care, Bad Homburg, Germany) at a dosage of 2.5 mg/kg for 4 days was started during surgery and continued until day 3. Before revascularisation, 500 mg of methylprednisolone was given intravenously. An additional 250 mg of methylprednisolone was given on day 1 and 125 mg on day 2. Steroids were then switched to oral prednisolone and tapered rapidly to 25 mg on day 8. Prednisolone was further reduced to 7.5 mg at 1 year. Tacrolimus (Fujisawa, Munich, Germany) was started at a dose of 0.20 mg/kg body weight in 2 oral doses and then adjusted to maintain levels of 15 ng/ml during the first month after surgery, 12 ng/ml between months 2 and 6 and 10 ng/ml thereafter. In addition, the patient was given 1 g of mycophenolate mofetil (MMF) twice a day (Roche, Basel, Switzerland). Maculopapulous lesions typical of rejection of the skin became apparent on day 55 and were treated successfully with 750 mg and 2 doses of 500 mg methylprednisolone and topical tacrolimus and steroid ointment. After 30 months, when graft function had reached a high level, immunosuppression (IS) was changed according to a previously designed protocol: steroids were withdrawn, and rapamycin started at 2 mg/day, aiming for trough levels of 4 8 g/ml. Simultaneously, tacrolimus was reduced to trough levels of 3 4 g/ml. Over the following 3 months, tacrolimus was slowly tapered and then discontinued, leaving the patient on rapamycin and MMF. Method As human bone biopsies are difficult to obtain, bone healing was assessed by ultrasound and radiography. Onset and course of early blood vessel ingrowth, development of soft tissue callus and late ossified callus formation were investigated at the osteotomy sites. Homogeneous union was defined as uniform bone structure on all projections; missing union was defined as radiolucency at the osteotomy site without calcification and was differentiated from a calcified filling called hard callus. Stability of the forearm bones was determined by radiological signs of hardware loosening. The type of bone healing was classified on radiographs according to Burchardt, as follows: type I, bone healing identical to autografts with remodelling and incorporation of the graft and no fatigue failure; type II, chronic repair with delayed union or nonunion, peripheral resorption with loss of graft size, internal resorption and decrease in mechanical strength; type III, no healing and complete graft resorption [1].

4 274 M. Gabl, S. Pechlaner, M. Lutz et al. Results Vascular invasion and early callus formation were visible by colour Doppler ultrasound at week 3. Vessels approached the osteotomy sites from the median side where the periosteal flap was positioned. At week 7, soft tissue callus formation was identified by grey-scale ultrasound. Hard callus of the forearm bone first appeared at month 4. Osseous union was observed between month 7 and month 11. At 1 year, homogeneous osseous union of the 4 forearm bones was terminated. All grafted bones were incorporated fully without any signs of chronic healing. The grafts showed healing Type 1. There were no signs of instability, with no loosening of the hardware devices in either donor or recipient bone. spared-off plate hole. There was no fracture sign proximal or distal of the plate. On 15 March 2005, the radiolucent gap was visible in posteroanterior and lateral radiographs. On 6 June 2005, the fracture gap remained radiolucent with signs of calcified filling and a beginning of appositional bone formation. As rapamycin was suspected to slow bone healing, dosage was reduced to achieve trough levels of 2 4 ng/ml and tacrolimus restarted (trough levels 3 5 ng/ml). After the fracture had healed completely, on 9 September 2005, tacrolimus was withdrawn and rapamycin again increased to achieve serum trough levels of 4 8 ng/ml. In addition, splinting and low-pulsed ultrasound was used to improve bone healing. Time course to bone union was delayed and was achieved at month 8. Fracture Healing The same patient sustained a distal radius fracture at his left wrist on November 2003 during a motorcycle accident while travelling in South America. He first showed up for X-ray control at our hospital on 2 December The fracture was treated conservatively by splinting. The radial styloid fracture fragment was dislocated and appeared as a radiolucent fracture line. The metaphyseal area was compressed, the intraarticular fracture pattern showed no significant steps. On 14 January 2004, some radiolucency at the metaphyseal area was visible, with beginning radial appositional bone formation. In March 2004, the metaphyseal compression was hyperdense and showed trabeculae bridging the fracture fragments. At that time, immunosuppression consisted of rapamycin (serum trough levels 4 8 ng/ml) and MMF (2 g). Time course of this metaphyseal, cancellous bone healing was delayed compared with normal fracture healing, which may have been caused by the target of rapamycin (ToR) inhibitor he was taking. In January 2005, the patient fell while walking on a snow-covered footpath and sustained a fracture of his left radius. The fracture line at the shaft was at the former osteotomy site. The plate showed slight bending without loosening of the screws. The frontal fracture was beneath a Discussion The biological process of bone healing in hand transplantation can adversely be influenced by instability at the site of osteotomy or by impairment of the vascular supply. From experimental studies, the vascular supply of the diaphysis of radius and ulna is known to be primarily supported by the palmar and dorsal interosseous artery. The nutrient foramina are mainly located at the interosseous margin and the palmar aspect of the radius, with the vessels intruding the bone from a periosteal network, which is supplied segmentally [15 17]. Following hand amputation, the level of osteotomy for rigid stabilisation by plates will be proximal to the distal fourth of the length of the forearm bones and so be in a poorly vascularised region. Soft tissue damage due to the initial trauma, and consecutive scarring and fibrosis can additionally impair vascularity and thus influence biological healing capacity. Apart from these critical biomechanical and biological aspects, the complex process of bone healing in hand transplantation is additionally impaired by immunological reactions and possibly by immunosuppressive medication. Bone healing in hand transplantation is healing of a vascularised, allogeneic cortical graft under immunosuppression. Immunology and the lack

5 Bone Healing in Hand Transplantation 275 of precise monitoring still leave many aspects of this biological process unknown. Stability Bone stability is essential for musculotendinous function and therefore a primary goal in rehabilitation programmes. Due to the immunogenicity, bone strength in hand transplantation remained a challenge in rehabilitation over the years. Close observations are required to calculate the risk of graft failure or fracture. Strategies applied to optimise early stable bone union in an undefined immunological environment are based on the principle of maximal stability at the osteotomy site and the idea to optimise the biological circumstances. Compression stimulates bone healing. Primary bone healing was intended in all patients and attempted to be achieved by different plate systems. No attempts have been reported to achieve union by indirect bone healing using intramedullary stabilisation or external fixation. The Lyon group [18] used 7-hole plates and 4.5-mm screws for fixation of both forearm bones in their two patients. Although rejection episodes occurred, bone healing was reported to be normal [19]. The forearm bones of the first Louisville patient were stabilised with 3.5-mm metal plates [20]. Two rejection episodes 6 and 20 weeks after surgery did not impair bone healing. In the patient presented in this chapter, 7- and 8-hole plates with 3.5-mm screws were used for bone stabilisation. Unfortunately, no further reports are available as of today about bone healing in the other recipients. All plate systems were stable enough to tolerate the individual rehabilitation protocols. No hardware failure or loosening was reported in the early postoperative period. To optimise local conditions at the osteotomy site, it was attempted to improve oxygenation at the fracture site or offer osteoinductive and osteoconductive elements. The French group used recipient cancellous bone chips from the iliac crest to support bone union. Radiographs at 3 months confirmed solid callus formation. Nonvascularised autologous cancellous bone grafts under sufficient immunosuppression showed solid healing at the donor recipient bone junction. To improve bone healing in the early period of repair, a vascularised periosteal flap from the recipient to cover the osteotomy sites was used in the Austrian patient [21]. The fact that the onset of callus formation with first signs of vascular ingrowth occurred where the local periosteal flap was positioned may be evidence that this strategy was helpful. Time Course of Bone Healing The time course of bone healing in sufficiently immunosuppressed patients with hand transplantation is delayed compared with nonimmunosuppressed patients but similar to that seen after replantation. Despite their known effect on bone metabolism, glucocorticoids seem not to delay bone healing compared with the time course in replantation. In the Austrian patient the time course of bone healing was not affected by a rejection episode even though the onset of antirejection treatment was delayed a few days because of misinterpretation of skin histology. Also, repeated acute rejections, as reported by other centres, showed no negative impact on bone healing. In our patient, primary bone healing was achieved at month 11. Bone bridging by the use of autologous cancellous grafts can be achieved at month 3, as reported by the French group [18]. Bone Strength Unfortunately, there is no parameter to estimate bone strength by radiological means. The composition of necrotic and new bone in cancellous bone grafts, as we observed in the distal radius fracture, and the porosity of creeping substitution, as we had to deal with in the radius shaft fracture, remains largely unknown. The use of primary bone grafting for the treatment of forearm fractures is not obligatory. However, in hand transplantation with prolonged intervals of primary bone healing, additional bone grafting can improve bone union and probably bone strength at the osteotomy site.

6 276 M. Gabl, S. Pechlaner, M. Lutz et al. In our very active patient, we had to deal with the problem of fracture healing under pharmacological immunosuppression. Though fractures in immunosuppressed patients are not rare, fracture healing in transplanted donor hands has not been reported hitherto. Union was delayed after both fractures of the distal radius metaphysis and the cortical radial shaft, respectively. Union of the distal radius metaphysis took 3 months, which is comparable to the bridging of the cancellous bone grafts reported in the French patient. The shaft fracture demonstrates the dilemma of estimating bone strength radiologically even after 5 years. In united bone fragments with a plate in place, a fracture usually occurs at the perimeter of the hardware, as the locus of minoris resistentiae. In our patient, these locations seemed to be more stable than the primary osteotomy site, which is still supported by the plate. Thus, active hand recipients should be informed about the impaired stability of the osseous components of their graft. Immunosuppression and Bone Healing Immunosuppression was based on tacrolimus in all patients and included steroids and MMF. Tacrolimus is reported to induce alkaline phosphatase, a marker of osteoblast activity, and also to enhance osteoblastic differentiations induced by bone morphogenic protein (BMP)-4 [22]. The proper use of tacrolimus is essential to lower the dose of glucocorticoids, thereby positively influencing bone mass evolution [23]. Early callus formation and early revascularisation imply that immunosuppression had no adverse effect on vascular ingrowth. Not only the initial phase of the bone healing process but also the disappearance of vascular ingrowth and callus maturation to collagen and ossified structures were comparable to bone healing after replantation. Thus, immunosuppressive drugs used so far after hand transplantation seem not to impair cellular biology during maturation of soft tissue callus to chondral and ossified callus. No direct influence on bone healing by the applied immunosuppressive regimes or by early rejection episodes was observed on ultrasound or radiographs. Whether or not rapamycin delays fracture healing remains to be studied. Conclusion The primary goal of forearm bone reconstruction in hand transplantation is to achieve and maintain stability enabling early motion rehabilitation. In all reported hand transplants, bone union was achieved by direct bone healing using various plate systems. Biology at the osteotomy site was in some patients supported by primary bone grafting and periosteal flaps, which seemed to be beneficial. The time course of bone union at the osteotomy site was equivalent to replantation and delayed in comparison to normal fracture healing. Immunosuppression was based on tacrolimus in all patients and included steroids and MMF and did not adversely influence bone healing. Time course of bone healing following fracture was delayed compared with normal fracture healing. Bone strength at the osteotomy site seems to remain reduced over 5 years. Acknowledgement The Authors thank the following staff members of the Innsbruck University Hospital for their support and helpful comments: P. Angermann, H. Hussl, M. Ninkovic, R. Zimmermann, (members of the surgical team), G. Bodner (radiologist) References 1. Burchardt H (1983) The biology of bone graft repair. Clin Orthop 174: Ostrum RF, Chao EY, Bassett CA et al (1994) Bone injury, regeneration and repair. In: Sheldon RS (ed) Orthopaedic Basis. Science.American Academy of Orthopedic Surgeons, pp Enneking WF, Eady JL, Burchardt H (1980) Autogenous cortical bone grafts in the reconstruction of segmental skeletal defects. J Bone Joint Surg Am 62: Schafer D, Jager K, Fricker R et al (1997) Quantitative monitoring of blood supply to knee joint transplants in dogs. Eur Surg Res 29:

7 Bone Healing in Hand Transplantation Bensusan JS, Davy DT, Goldberg VM et al (1991) The effects of vascularity and cyclosporin A on the mechanical properties of canine fibular autografts. J Biomech 25: Innis PC, Randolph MA, Paskert JP et al (1991) Related articles.vascularized bone allografts: in vitro assessment of cell-mediated and humoral responses. Plast Reconstr Surg 87: Doi K, DeSantis G, Singer DI et al (1989) The effect of immunosuppression on vascularised allografts.a preliminary report. J Bone Joint Surg Br 71: Mathes DW, Randolph MA, Bourget JL et al (2002) Recipient bone marrow engraftment in donor tissue after long-term tolerance to a composite tissue allograft. Transplantation 73: Kirschner MH, Brauns L, Gonschorek O et al (2000) Vascularised knee joint transplantation in man: the first two years experience. Eur J Surg 166: Thompson RC Jr, Pickvance EA, Garry D (1993) Fractures in large-segment allografts. J Bone Joint Surg Am 75: Lee WP, Yaremchuk MJ, Pan YC et al (1991) Relative antigenicity of components of a vascularized limb allograft. Plast Reconstr Surg 87: Muramatsu K, Doi K,Akino T et al (1997) Longer survival of rat limb allograft. Combined immunosuppression of FK-506 and 15-deoxyspergualin. Acta Orthop Scand 68: Gabl M, Pechlaner S, Lutz M et al (2004) Bilateral hand transplantation: bone healing under immunosuppression with tacrolimus, mycophenolate mofetil, and prednisolone. J Hand Surg [Am] 29: Margreiter R, Brandacher G, Ninkovic M et al (2002) A double-hand transplant can be worth the effort! Transplantation 74: Menck J, Schreiber HW, Hertz T, Burgel N (1994) Angioarchitecture of the ulna and radius and their practical relevance. Langenbecks Arch Chir 379: Giebel GD, Meyer C, Koebke J, Giebel G (1997) Arterial supply of forearm bones and its importance for the operative treatment of fractures. Surg Radiol Anat 19: Wright TW, Glowczewskie F (1998) Vascular anatomy of the ulna. J Hand Surg [Am] 23: Dubernard JM,Owen E,Herzberg G et al (1999) Human hand allograft report on first 6 months. Lancet 17: Petruzzo P, Revillard JP, Kanitakis J et al (2003) First human double hand transplantation efficacy of a conventional immunosuppressive protocol. Clin Transplant 17: Jones JW, Gruber SA, Barker JH, Breidenbach WC (2002) Successful hand transplantation. One-year follow up. N Engl J Med 7: Piza-Katzer H, Ninkovic N, Pechlaner S et al (2002) Double hand transplantation functional outcome after 18 months. J Hand Surg [Br] 27: Tang L, Ebara S, Kawasaki S et al (2002) FK506 enhanced osteoblastic differentiation in mesenchymal cells. Cell Biol Int 26: Monegal A, Navasa M, Guanabens N et al (2001) Bone mass and mineral metabolism in liver transplant patients treated with FK506 or cyclosporine A. Calcif Tissue Int 68:83 86