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Comparison of frozen and freeze-dried particulate bone allografts
Cryobiology 55 (2007) 167–170 www.elsevier.com/locate/ycryo
Brief Communication
Comparison of frozen and freeze-dried particulate bone allografts
q
Theodore Malinin *, H. Thomas Temple Tissue Bank, Department of Orthopaedics (R-12), University of Miami, Miller School of Medicine, P.O. Box 01696, Miami, FL 33101, USA The Mannheimer Foundation, Homestead, FL, USA Received 27 March 2007; accepted 23 May 2007 Available online 15 June 2007
Abstract Freeze-dried and frozen particulate bone allografts are used interchangeably on the assumption that the biologic behavior of these grafts is similar. Dissimilarities in biologic behavior and differences in the rate and extent of bone incorporation of freeze-dried and frozen particulate grafts were demonstrated in a comparative study using a non-human primate model. Freeze-dried particulate allografts induced new bone formation and healing of the osseous defects much faster than the frozen allografts. 2007 Elsevier Inc. All rights reserved. Keywords: Freeze-drying; Freezing; Bone allografts; Non-human primates
The principal object of bone grafting is to heal bone defects. The rapidity of healing is important. The sooner the bone heals the sooner the patient can return to normal activities. Active allogeneic or autologous bone grafts induce vascularization, mobilize osteoprogenitor cells from the mesenchymal tissue of the host and rapidly convert these into osseous tissues [10]. Autologous bone grafts are usually transplanted fresh, while allografts are subjected to preservation and hence are of interest to cryobiologists. The two techniques commonly used for preservation of bone allografts are freezing and freeze-drying [5–7,9]. Although the processes are distinct, each with its advantages and disadvantages, both frozen and freeze-dried bone allografts are distributed by the many tissue banks, with surgeons being told there is little difference between the two. This may not be correct. No studies are reported in the literature in which biologic behavior of frozen and freeze-dried particulate bone allografts is compared under identical conditions. Therefore, a study comparing frozen and freeze-dried alloq This study was supported by Kacklinsky Foundation, Mannheimer Foundation and the Department of Orthopaedics, University of Miami. * Corresponding author. Fax: +1 305 243 4622. E-mail address: [email protected] (T. Malinin).
0011-2240/$ - see front matter 2007 Elsevier Inc. All rights reserved. doi:10.1016/j.cryobiol.2007.05.007
grafts in a non-human primate model was performed. Reports of studies comparing frozen and freeze-dried structural allografts, have produced divergent information not applicable to the present study. Experiments were performed on one extremity of 9 young, outbred baboons (Papio heamadryas). The animal study was approved by the Institutional Animal Care and Use Committee. Each animal had defects made in the distal femur and when possible in the proximal tibia according to the method described by Maatz et al. [4]. The defects were 9 mm in diameter and 15 mm deep. Three defects were left open (control), three were filled with freeze-dried microparticulate bone, and three with rapidly frozen microparticulate bone (diameter of particles 90–300 l). The size of freeze-dried bone was identical to that of frozen bone. Three additional defects were filled with autologous bone and served as positive controls. Bone for preparation of particulate allografts was excised aseptically from intact lower limbs of animals used in other experiments, cleaned of soft tissues, cut into pieces, washed with saline, wrapped in cotton towels, and frozen in liquid nitrogen vapor. To be freeze-dried, bone was removed from liquid nitrogen and placed on the shelves of the freeze-dryer precooled to 30 C. The freeze-dryer chamber was then sealed with a vacuum at 30 mTorr or less
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T. Malinin, H.T. Temple / Cryobiology 55 (2007) 167–170
with condenser temperatures of 50–60 C. Bone specimens placed on the shelves of the freeze-dryer from liquid nitrogen warmed up to the chamber temperatures within 30 min. Bone was freeze-dried for 5 days to a residual moisture of 3–5%. Overdrying was avoided. This freeze-drying technique has been previously described [6]. Freeze-dried bone segments were ground incrementally in industrial grinders. During the grinding, temperature was monitored with a thermocouple attached to the wall of the grinder chamber. The temperature was not allowed to rise above 50 C. Ground, freeze-dried bone was sieved through USA Standard Testing sieves and particles of 90– 300 l were retained. This specific particle size was shown to be effective in achieving bone healing in a non-human primate model [7]. Frozen bone particles were prepared similarly except that frozen rather than freeze-dried bone was subjected to grinding. Bone was kept frozen by the addition of liquid nitrogen throughout the grinding and sieving processes. Bone prepared from each animal was kept segregated, and packaged into 5 g aliquotes. Two experimental animals from each group were sacrificed at 6 weeks. The third animal was sacrificed at 12 weeks. Bones containing defects were frozen in dry ice, radiographed, sectioned with a rotary diamond saw and photographed. Representative pieces were fixed in 10% formalin—Earle’s balanced salt solution, decalcified, dehydrated through graded alcohols, cleared in xylene, and
embedded in paraffin. Sections were cut at 5 lm and stained with hematoxylin and eosin and Masson’s trichrome stain. The results were obtained by combining gross appearance, radiographic appearance, and histomorphometric measurements of the defect. The latter were performed by calculating the percentage of new bone occupying the defects in cross sections using a Image Pro-Plus, 5.0 Media Cybernetics, Inc., Silver Spring, MD. Although the number of animals used in this experiment was small the results were clear cut. At 6 weeks defects filled with freeze-dried particulate bone were completely healed with obliterated cavities filled with newly formed bone (Fig. 1). Defects filled with frozen bone remained unhealed. On gross examination, frozen bone allograft was clearly recognizable within the defect. New bone formation was present only in the periphery of the defect with new bone replacing less than 20% of the defect. Interspersed mesenchymal cells and fibro-osseous tissue was observed between the graft and host bone. Intense osteoblastic activity was present only in the adjacent host bone (Fig. 2). Defects filled with autografts were completely healed and were morphologically and microscopically similar to the freeze-dried microparticulate graft-filled defects. Control (unfilled) defects remained open with new bone formation present only in the periphery. In 12 week specimens defects filled with frozen and freeze-dried allografts as well as autograft bone were completely healed. However, in the histologic sections
Fig. 1. Freeze-dried particulate cortical bone allograft was used to fill defect in the proximal tibia of a baboon. Proximal tibia was examined 6 weeks posttransplantation. (a) Radiograph shows healed defect (arrows). (b) Gross specimen shows new bone formation in the defect (arrows). (c) Histological section shows the defect to be filled with newly formed cancellous bone. Host–graft junction is indicated by arrows. H & E ·25. (d) Osteoblastic activity is prominent in the newly formed bone. H & E ·100.
T. Malinin, H.T. Temple / Cryobiology 55 (2007) 167–170
169
Fig. 2. Frozen particulate bone was used to fill a defect in proximal tibia of a baboon. Proximal tibia was examined 6 weeks post-transplantation. (a) Lateral and anteroposterior radiographs show clearly recognizable defects (arrows). (b) Gross specimen shows virtually unchanged allograft filling the defect. (c) A narrow rim of newly formed bone is present in the defect’s periphery. H & E ·100. (d) A rim of mesenchymal tissue surrounds the bone graft. H & E ·100.
remnants of the frozen bone allografts were still recognizable. Unfilled, control defects were replaced with new bone in less than 50% of the defect. The present experiments show distinct differences in the biology of freeze-dried and liquid nitrogen frozen particulate cortical bone allograft. Freezing at liquid nitrogen temperatures was chosen because according to the reports in the literature it affords the best method of freeze-preservation in as much as no ice crystal growth takes place at cryogenic temperatures [10]. Freeze-dried allograft particles incorporate rapidly. On the other hand, incorporation and replacement of frozen particulate allograft was delayed compared to the freeze-dried allograft and autograft-filled defects. The reason for the differences might be attributable to the immunogenicity of these grafts. The processes of freezing and freeze-drying reduce immunogenicity of bone allografts, but to a greater extent in the freeze-dried preparations [2,11,12]. Rapidity of healing of bone defects is important clinically. Once bone is healed patients can return to normal activities. Initially, in the latter half of the last century most transplanted bone allografts were freeze-dried [1,3,5]. However, subsequent to successful transplantation of large frozen allografts [8,9] transplantation of frozen bone allografts came into vogue. This seems to have occurred without accompanying laboratory and clinical studies. Results of the present experiments demonstrate the superiority of freeze-dried particulate cortical bone
allograft over its frozen counterpart. Of course, these results are applicable only to particulate allografts. These findings are of practical importance, but it must be borne in mind that the findings were derived from a small number of experimental animals, and may be limited to particulate cortical bone allografts. Further studies are needed to determine the differences of bone incorporation in larger structural freeze-dried grafts compared to their frozen counterparts. Data currently available on this subject is controversial. References [1] C.R. Carr, G.W. Hyatt, Clinical evaluation of freeze-dried bone grafts, J. Bone Joint Surg. 37 (1955) 549–552. [2] A.A. Czitrom, T. Axelrod, B. Fernandez, Antigen presenting cells and bone allotransplantation, Clin. Orthop. 197 (1985) 27–31. [3] R.B. Gresham, Freeze-drying of human tissues for clinical use, Cryobiology 1 (1964) 150–156. [4] R. Maatz, W. Lentz, R. Graf, Spongiosa test of bone graft for transplantation, J. Bone Joint Surg. Am. 88 (2006) 762– 770. [5] T.I. Malinin, C.B. Thompson, M.D. Brown, Freeze-dried tissue allografts in surgery, in: A.M. Karow, D.E. Pegg (Eds.), Organ Preservation for Transplantation, Marcel Dekker, New York and Basel, 1981, pp. 667–689. [6] T.I. Malinin, Preparation and banking of bone and tendon allografts, in: M.B. Habal, A.H. Reddi (Eds.), Bone Grafts and Bone Substitutes, WB Saunders, Philadelphia, 1992, pp. 206–225. [7] T.I. Malinin, Principles of musculoskeletal tissue banking and transplantation, in: F. Pietrzak, B. Eppley (Eds.), Musculoskeletal
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T. Malinin, H.T. Temple / Cryobiology 55 (2007) 167–170
Tissue Regeneration: Biologic Materials and Methods. Humana Press, Totowa, NJ (in press). [8] H.J. Mankin, S. Doppelts, W.W. Tomford, Clinical experience with allograft implantation: the first ten years, Clin. Orthop. 174 (1993) 69. [9] W. Mnaymneh, T.I. Malinin, R.D. Lackman, F.J. Hornicek, L. Ghandur-Mnaymneh, Massive distal femoral osteoarticular allografts
after resection of bone tumors, Clin. Orthop. 303 (1994) 103–115. [10] A.P. Rinfret, Thermal history, Cryobiology 2 (1966) 171–180. [11] S. Stevenson, S.E. Emery, V.M. Goldberg, Factors affecting bone graft incorporation, Clin. Orthop. 324 (1996) 66–74. [12] S. Stevenson, Biology of bone grafts, Orthop. Clin. North Am. 30 (1999) 543–552.
Brief Communication
Comparison of frozen and freeze-dried particulate bone allografts
q
Theodore Malinin *, H. Thomas Temple Tissue Bank, Department of Orthopaedics (R-12), University of Miami, Miller School of Medicine, P.O. Box 01696, Miami, FL 33101, USA The Mannheimer Foundation, Homestead, FL, USA Received 27 March 2007; accepted 23 May 2007 Available online 15 June 2007
Abstract Freeze-dried and frozen particulate bone allografts are used interchangeably on the assumption that the biologic behavior of these grafts is similar. Dissimilarities in biologic behavior and differences in the rate and extent of bone incorporation of freeze-dried and frozen particulate grafts were demonstrated in a comparative study using a non-human primate model. Freeze-dried particulate allografts induced new bone formation and healing of the osseous defects much faster than the frozen allografts. 2007 Elsevier Inc. All rights reserved. Keywords: Freeze-drying; Freezing; Bone allografts; Non-human primates
The principal object of bone grafting is to heal bone defects. The rapidity of healing is important. The sooner the bone heals the sooner the patient can return to normal activities. Active allogeneic or autologous bone grafts induce vascularization, mobilize osteoprogenitor cells from the mesenchymal tissue of the host and rapidly convert these into osseous tissues [10]. Autologous bone grafts are usually transplanted fresh, while allografts are subjected to preservation and hence are of interest to cryobiologists. The two techniques commonly used for preservation of bone allografts are freezing and freeze-drying [5–7,9]. Although the processes are distinct, each with its advantages and disadvantages, both frozen and freeze-dried bone allografts are distributed by the many tissue banks, with surgeons being told there is little difference between the two. This may not be correct. No studies are reported in the literature in which biologic behavior of frozen and freeze-dried particulate bone allografts is compared under identical conditions. Therefore, a study comparing frozen and freeze-dried alloq This study was supported by Kacklinsky Foundation, Mannheimer Foundation and the Department of Orthopaedics, University of Miami. * Corresponding author. Fax: +1 305 243 4622. E-mail address: [email protected] (T. Malinin).
0011-2240/$ - see front matter 2007 Elsevier Inc. All rights reserved. doi:10.1016/j.cryobiol.2007.05.007
grafts in a non-human primate model was performed. Reports of studies comparing frozen and freeze-dried structural allografts, have produced divergent information not applicable to the present study. Experiments were performed on one extremity of 9 young, outbred baboons (Papio heamadryas). The animal study was approved by the Institutional Animal Care and Use Committee. Each animal had defects made in the distal femur and when possible in the proximal tibia according to the method described by Maatz et al. [4]. The defects were 9 mm in diameter and 15 mm deep. Three defects were left open (control), three were filled with freeze-dried microparticulate bone, and three with rapidly frozen microparticulate bone (diameter of particles 90–300 l). The size of freeze-dried bone was identical to that of frozen bone. Three additional defects were filled with autologous bone and served as positive controls. Bone for preparation of particulate allografts was excised aseptically from intact lower limbs of animals used in other experiments, cleaned of soft tissues, cut into pieces, washed with saline, wrapped in cotton towels, and frozen in liquid nitrogen vapor. To be freeze-dried, bone was removed from liquid nitrogen and placed on the shelves of the freeze-dryer precooled to 30 C. The freeze-dryer chamber was then sealed with a vacuum at 30 mTorr or less
168
T. Malinin, H.T. Temple / Cryobiology 55 (2007) 167–170
with condenser temperatures of 50–60 C. Bone specimens placed on the shelves of the freeze-dryer from liquid nitrogen warmed up to the chamber temperatures within 30 min. Bone was freeze-dried for 5 days to a residual moisture of 3–5%. Overdrying was avoided. This freeze-drying technique has been previously described [6]. Freeze-dried bone segments were ground incrementally in industrial grinders. During the grinding, temperature was monitored with a thermocouple attached to the wall of the grinder chamber. The temperature was not allowed to rise above 50 C. Ground, freeze-dried bone was sieved through USA Standard Testing sieves and particles of 90– 300 l were retained. This specific particle size was shown to be effective in achieving bone healing in a non-human primate model [7]. Frozen bone particles were prepared similarly except that frozen rather than freeze-dried bone was subjected to grinding. Bone was kept frozen by the addition of liquid nitrogen throughout the grinding and sieving processes. Bone prepared from each animal was kept segregated, and packaged into 5 g aliquotes. Two experimental animals from each group were sacrificed at 6 weeks. The third animal was sacrificed at 12 weeks. Bones containing defects were frozen in dry ice, radiographed, sectioned with a rotary diamond saw and photographed. Representative pieces were fixed in 10% formalin—Earle’s balanced salt solution, decalcified, dehydrated through graded alcohols, cleared in xylene, and
embedded in paraffin. Sections were cut at 5 lm and stained with hematoxylin and eosin and Masson’s trichrome stain. The results were obtained by combining gross appearance, radiographic appearance, and histomorphometric measurements of the defect. The latter were performed by calculating the percentage of new bone occupying the defects in cross sections using a Image Pro-Plus, 5.0 Media Cybernetics, Inc., Silver Spring, MD. Although the number of animals used in this experiment was small the results were clear cut. At 6 weeks defects filled with freeze-dried particulate bone were completely healed with obliterated cavities filled with newly formed bone (Fig. 1). Defects filled with frozen bone remained unhealed. On gross examination, frozen bone allograft was clearly recognizable within the defect. New bone formation was present only in the periphery of the defect with new bone replacing less than 20% of the defect. Interspersed mesenchymal cells and fibro-osseous tissue was observed between the graft and host bone. Intense osteoblastic activity was present only in the adjacent host bone (Fig. 2). Defects filled with autografts were completely healed and were morphologically and microscopically similar to the freeze-dried microparticulate graft-filled defects. Control (unfilled) defects remained open with new bone formation present only in the periphery. In 12 week specimens defects filled with frozen and freeze-dried allografts as well as autograft bone were completely healed. However, in the histologic sections
Fig. 1. Freeze-dried particulate cortical bone allograft was used to fill defect in the proximal tibia of a baboon. Proximal tibia was examined 6 weeks posttransplantation. (a) Radiograph shows healed defect (arrows). (b) Gross specimen shows new bone formation in the defect (arrows). (c) Histological section shows the defect to be filled with newly formed cancellous bone. Host–graft junction is indicated by arrows. H & E ·25. (d) Osteoblastic activity is prominent in the newly formed bone. H & E ·100.
T. Malinin, H.T. Temple / Cryobiology 55 (2007) 167–170
169
Fig. 2. Frozen particulate bone was used to fill a defect in proximal tibia of a baboon. Proximal tibia was examined 6 weeks post-transplantation. (a) Lateral and anteroposterior radiographs show clearly recognizable defects (arrows). (b) Gross specimen shows virtually unchanged allograft filling the defect. (c) A narrow rim of newly formed bone is present in the defect’s periphery. H & E ·100. (d) A rim of mesenchymal tissue surrounds the bone graft. H & E ·100.
remnants of the frozen bone allografts were still recognizable. Unfilled, control defects were replaced with new bone in less than 50% of the defect. The present experiments show distinct differences in the biology of freeze-dried and liquid nitrogen frozen particulate cortical bone allograft. Freezing at liquid nitrogen temperatures was chosen because according to the reports in the literature it affords the best method of freeze-preservation in as much as no ice crystal growth takes place at cryogenic temperatures [10]. Freeze-dried allograft particles incorporate rapidly. On the other hand, incorporation and replacement of frozen particulate allograft was delayed compared to the freeze-dried allograft and autograft-filled defects. The reason for the differences might be attributable to the immunogenicity of these grafts. The processes of freezing and freeze-drying reduce immunogenicity of bone allografts, but to a greater extent in the freeze-dried preparations [2,11,12]. Rapidity of healing of bone defects is important clinically. Once bone is healed patients can return to normal activities. Initially, in the latter half of the last century most transplanted bone allografts were freeze-dried [1,3,5]. However, subsequent to successful transplantation of large frozen allografts [8,9] transplantation of frozen bone allografts came into vogue. This seems to have occurred without accompanying laboratory and clinical studies. Results of the present experiments demonstrate the superiority of freeze-dried particulate cortical bone
allograft over its frozen counterpart. Of course, these results are applicable only to particulate allografts. These findings are of practical importance, but it must be borne in mind that the findings were derived from a small number of experimental animals, and may be limited to particulate cortical bone allografts. Further studies are needed to determine the differences of bone incorporation in larger structural freeze-dried grafts compared to their frozen counterparts. Data currently available on this subject is controversial. References [1] C.R. Carr, G.W. Hyatt, Clinical evaluation of freeze-dried bone grafts, J. Bone Joint Surg. 37 (1955) 549–552. [2] A.A. Czitrom, T. Axelrod, B. Fernandez, Antigen presenting cells and bone allotransplantation, Clin. Orthop. 197 (1985) 27–31. [3] R.B. Gresham, Freeze-drying of human tissues for clinical use, Cryobiology 1 (1964) 150–156. [4] R. Maatz, W. Lentz, R. Graf, Spongiosa test of bone graft for transplantation, J. Bone Joint Surg. Am. 88 (2006) 762– 770. [5] T.I. Malinin, C.B. Thompson, M.D. Brown, Freeze-dried tissue allografts in surgery, in: A.M. Karow, D.E. Pegg (Eds.), Organ Preservation for Transplantation, Marcel Dekker, New York and Basel, 1981, pp. 667–689. [6] T.I. Malinin, Preparation and banking of bone and tendon allografts, in: M.B. Habal, A.H. Reddi (Eds.), Bone Grafts and Bone Substitutes, WB Saunders, Philadelphia, 1992, pp. 206–225. [7] T.I. Malinin, Principles of musculoskeletal tissue banking and transplantation, in: F. Pietrzak, B. Eppley (Eds.), Musculoskeletal
170
T. Malinin, H.T. Temple / Cryobiology 55 (2007) 167–170
Tissue Regeneration: Biologic Materials and Methods. Humana Press, Totowa, NJ (in press). [8] H.J. Mankin, S. Doppelts, W.W. Tomford, Clinical experience with allograft implantation: the first ten years, Clin. Orthop. 174 (1993) 69. [9] W. Mnaymneh, T.I. Malinin, R.D. Lackman, F.J. Hornicek, L. Ghandur-Mnaymneh, Massive distal femoral osteoarticular allografts
after resection of bone tumors, Clin. Orthop. 303 (1994) 103–115. [10] A.P. Rinfret, Thermal history, Cryobiology 2 (1966) 171–180. [11] S. Stevenson, S.E. Emery, V.M. Goldberg, Factors affecting bone graft incorporation, Clin. Orthop. 324 (1996) 66–74. [12] S. Stevenson, Biology of bone grafts, Orthop. Clin. North Am. 30 (1999) 543–552.