Cryopreservation with dimethyl sulfoxide sustains partially the biological function of osteochondral tissue

Bone 33 (2003) 352–361

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Cryopreservation with dimethyl sulfoxide sustains partially the biological function of osteochondral tissue R.J. Egli,a,b,* A. Sckell,a,b C.R. Fraitzl,a,b R. Felix,b R. Ganz,a W. Hofstetter,b and M. Leuniga a

Department of Orthopedic Surgery, University of Berne, Inselspital, Berne, Switzerland b Department of Clinical Research, University of Berne, Berne, Switzerland Received 1 April 2003; accepted 4 May 2003

Abstract The clinical routine use of bone allograft transplants dates back to the discovery that grafts devitalized by freezing bear a reduced antigenicity. Graft failures, caused by a host versus graft reaction, however, remain a clinical problem. Previous investigations on pancreatic islet allografts revealed improved survival and biological function when fast cryopreservation (⫺70°C/min) was performed in the presence of dimethyl sulfoxide (DMSO). The aim of this study was to determine the effect of fast freezing using DMSO on the biological function of osteochondral tissues. Organ culture was performed with neonatal femora of mice, untreated, rapidly frozen (⫺70°C/min) with DMSO, or frozen without DMSO. After the culture, tissue morphology, cellular proliferation, osteoblast function, osteoclasts, and the presence of antigen-presenting cells were investigated. In untreated control femora histology appeared normal and proliferating and collagensynthesizing osteoblasts, osteoclasts, and B-cells and macrophages were present. In frozen femora (with and without DMSO) a disintegration of the periosteum and the epiphyseal growth plate were observed and no active osteoblasts could be detected. Osteoclasts were partially detached from the bone surface. Cell proliferation was fully blocked in femora frozen in the absence of DMSO, while freezing in the presence of DMSO preserved cell proliferation in the medullary canal. The proliferating cells do not express epitopes present on the cells of the B-cell or macrophage lineages. Although the biological function of osteoblasts and osteoclasts was lost upon freezing of osteochondral tissue, DMSO included in freezing protocols preserves some residual cell viability which may be of importance for early graft revascularization as has been previously demonstrated by our group. © 2003 Elsevier Inc. All rights reserved. Keywords: Bone markers; Bone histology; Bone marrow

Introduction Allogeneic bone transplantation will remain an indispensable option in the limb preserving surgery of skeletal defects as long as biomaterial research and tissue engineering do not overcome current limitations inherent to artificial bone substitutes [1]. Despite the routine use of bone transplantation in clinics, grafting of allogeneic bone from the bone bank is frequently accompanied by complications, which seem to be caused primarily by host versus graft

* Corresponding author. Department of Clinical Research, Murtenstrasse 35, CH-3010 Berne, Switzerland. Fax: ⫹41-31-632-3297. E-mail address: [email protected] (R.J. Egli). 8756-3282/$ – see front matter © 2003 Elsevier Inc. All rights reserved. doi:10.1016/S8756-3282(03)00192-3

reactions (reviewed by Mankin et al. [2]). To avoid these reactions various strategies can be applied targeting either the recipient or the graft. Modulating the immune system of the recipients of the graft by immunosuppressive drugs is commonly used in the treatment of life-threatening diseases but is hardly justified in limb-threatening diseases as some of them are associated with significant side effects [2,3]. Therefore, for most orthopedic situations, targeting the graft rather than the host should be considered. In the past, slow freezing (⫺0.5 to ⫺2°C/min) had become the standard treatment for the preservation of bone allografts [4]. Beside the advantage of possible long-term storage this treatment reduces antigenicity by devitalizing the grafts [5] but does not alter initial graft biomechanics

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significantly [6]. Nevertheless, transplantation of frozen bone allografts is associated with significant local complications such as graft infections, fatigue fractures, and nonunions [7]. These complications mostly have been attributed to the remaining graft antigenicity [7] and to the devitalized grafts not being able to contribute to incorporation into the host tissue [8]. The strategy to improve the clinical outcome of allograft transplantation should reduce graft antigenicity while preserving the biological function. Various approaches to diminish antigenicity in different tissues and organs have been applied including antibodies against MHC complexes [9], tissue culture [10], cold and warm preservation [11], irradiation [12], freeze drying [12], and cryopreservation at low freezing rates [13]. The aim of these approaches consists of the selective destruction or inactivation of donor-derived antigen-presenting cells, such as dendritic cells, macrophages, and activated B-lymphocytes [14], which carry the major part of the graft antigenicity [15–17]. Previous investigations on pancreatic islets revealed improved survival and biological function of allografts when cryopreservation at a fast freezing rate (⫺70°C/min) was performed in the presence of the cryoprotectant dimethyl sulfoxide (DMSO) [18]. It was suggested that the applied protocol maintained the viability of insulin-producing cells, while dendritic cells, macrophages, and lymphocytes were destroyed due to the sensitivity of these cells to fast freezing [19]. So far, most research on bone allograft cryopreservation has been performed using slow or noncontrolled freezing without cryoprotective substances [5,20 –22]. The aim of the present study was to determine the effect of fast freezing with and without DMSO on osteochondral tissue in vitro. For this purpose, autoradiography for [methyl-3H]thymidine incorporation, in situ hybridization for mRNA encoding for the ␣1 chain of type I collagen (col ␣1(I)), enzyme histochemistry for tartrate-resistant acid phosphatase (TRAP), and immunohistochemistry for macrophages and B-lymphocytes were performed.

Material and methods Osteochondral tissue Intact femora of neonatal ddy mice not older than 24 h (1.6 –2.5 g) were used for this study. These neonatal femora have a mineralized bony diaphysis [23] and a cartilaginous epiphysis and were chosen since they are more active with respect to cell proliferation and matrix synthesis than adult tissue. Furthermore, they allow experiments using the dorsal skin-fold chamber to study angiogenesis and osteogenesis in vivo [24 –26]. Technically, after decapitation, both femora were dissected under aseptic conditions in phosphate-buffered saline (PBS; pH 7.4) at 4°C using a dissecting microscope. To determine the effect of cryopreservation in the pres-

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Fig. 1. Seven femora of neonatal mice were assigned to each of the three experimental groups: without pretreatment (Con), fast frozen in presence of DMSO (CryoDMSO; ⫺70°C/min), and fast frozen in absence of DMSO (Cryo; ⫺70°C/min).

ence and absence of DMSO, femora were assigned to three different groups (Fig. 1): fast frozen in the presence of DMSO (CryoDMSO; ⫺70°C/min; n ⫽ 7), fast frozen in the absence of DMSO (Cryo; ⫺70°C/min; n ⫽ 7), and without pretreatment (Con; n ⫽ 7). Cryopreservation Femora of the CryoDMSO group were exposed to increasing concentrations of DMSO at 4°C, according to the protocol by Menger et al. [27]: 0.67 M for 5 min, 1 M for 25 min, and 2 M for 15 min. Femora of Cryo were incubated in PBS at 4°C for 45 min prior to freezing. Thereafter, femora were placed in sterile cryotubes, cooled at a rate of ⫺70°C/min to ⫺70°C using a computer-controlled freezing device (Programmable Freezer Sypca 2010, Cryo Diffusion, N. Zivy and Cie S.A., Oberwil Basel, Switzerland), and stored at ⫺70°C for 24 h. To allow fast freezing, the tubes were not closed during the cooling phase, and the temperature probe was placed in an empty tube to control the temperature in the vicinity of the femora. Rapid thawing was performed by immersing the tubes in a 4°C water bath for 3 min. To remove DMSO, the femora were incubated in a solution containing 1 ml PBS and 1 ml 0.75 M sucrose for 30 min at 4°C and further washed with PBS. Meanwhile, frozen femora not treated with DMSO were incubated in PBS at 4°C. Organ culture Femora of all groups were cultured (24 h, 37°C, 5% CO2) in Dulbecco’s minimal essential medium (Oxoid AG, Basel, Switzerland) with 10% v/v fetal bovine serum (Oxoid AG, Basel, Switzerland) in 24-well plate tissue culture dishes. For the investigation of cellular proliferation, 7 ␮Ci/ml [methyl-3H]thymidine (Amersham International, Buckinghamshire, England) was in the culture medium during the 24-h culture period.

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Histological preparation After being cultured the femora were fixed in 4% paraformaldehyde/3% dextrane in PBS, decalcified for 4 days in 15% EDTA/0.5% paraformaldehyde/3% dextrane in PBS (pH 8.0), dehydrated in graded ethanol, cleared in xylol, and embedded in low-melting-point paraffin. Five-micrometer sections were stained with Mayer’s ha¨ malaun and eosin for histological analysis based on published protocols [28]. Autoradiography Sections were deparaffinized, dipped into Kodak NTB-2 photo emulsion (Kodak, Lausanne, Switzerland), and exposed for 72 to 96 h at 4°C. After development the sections were counterstained with Mayer’s ha¨ malaun and embedded with Eukitt (Inselspital-Apotheke, Berne, Switzerland). In situ hybridization for mRNA encoding for the ␣1 chain of type I collagen (col ␣1(I)) Digoxigenin-labeled riboprobes for mRNA encoding col ␣1(I) were synthesized as published [29]. Deparaffinized sections were hybridized with the riboprobes according to a protocol described previously [30] and detected by immunohistochemical analysis using an anti-digoxigenin antibody conjugated with alkaline phosphatase (Roche Diagnostics, Rotkreuz, Switzerland). Binding of the antibody was visualized with 5-bromo-4-chloro-3-indolyl phosphate and nitroblue tetrazolium (Sigma, Buchs, Switzerland). Thereafter, the sections were embedded with Aquamount (BDH Laboratory Supplies, Poole, England). Enzyme histochemistry for tartrate-resistant acid phosphatase TRAP, a marker for osteoclasts, was detected enzyme histochemically using a commercially available kit (leukocyte acid phosphatase; Sigma Diagnostics, St. Louis, MO, USA) [31]. Immunohistochemistry To characterize cells of the B-cell and macrophage lineages, immunohistochemistry for CD45R/B220- and F4/80-antigens, respectively, was performed on paraffin sections by an indirect peroxidase-conjugated streptavidin procedure [32]. Sections were deparaffinized and rehydrated in graded ethanol. Endogenous peroxidase was blocked with 0.3% H2O2 in absolute methanol for 2 ⫻ 15 min. Thereafter the

sections were washed in tris(hydroxymethyl)-aminomethanbuffered saline (TBS; pH 7.6) for 10 min and unspecific binding sites were blocked with normal sheep serum (50% v/v). The sections were then incubated with rat monoclonal antibodies (F4/80-antibodies [33]: hybridomas supernatant diluted 1:100 in TBS; CD45R/B220 antibodies (Becton– Dickinson, PharMingen, USA): diluted 1:250 in TBS) for 45 min at room temperature. Rat IgG was used as control. After being washed for 20 min in TBS, the sections were incubated with biotinylated sheep anti-rat IgG (diluted 1:1000 in TBS; Amersham Pharmacia Biotech Europe, Du¨ bendorf, Switzerland) for 45 min and, again after being washed with TBS, with streptavidin– horseradish peroxidase conjugate (diluted 1:2000 in TBS; Amersham Pharmacia Biotech Europe) for 45 min. Bound antibody was visualized using the peroxidase-specific substrate 3-amino-9ethylcarbazole. (Sigma Chemical, St. Louis, MO, USA). For the combination of immunohistochemistry and autoradiography, sections were covered with photo emulsion and exposed at 4°C (see autoradiography). The sections were finally embedded with Aquamount (BDH Laboratory Supplies). Results Morphology In untreated femora (Con) (Fig. 2A) morphology of the bony diaphysis and cartilaginous epiphysis appeared normal, as did the organization of the epiphyseal growth plate in proliferating and hypertrophic zones. The femora were surrounded by a continuous periosteum containing cells and extracellular matrix. Morphologically, the cell populations within the bone marrow were heterogenous with only a few disrupted cells. When femora were frozen with and without DMSO (CryoDMSO, Cryo) (Fig. 2B and C), morphology of the bony diaphysis and the cartilaginous epiphysis was comparable to the respective tissues in untreated femora with exception of the epiphyseal growth plate, where the functional organization was lost. Moreover, in frozen femora the periosteum appeared disrupted and partially absent with only a few cells left. Disorganization and necrosis in the bone marrow were observed in frozen femora, whereas in femora pretreated with DMSO (CryoDMSO) disruption of cells was less pronounced. Cell proliferation Autoradiography for [methyl-3H]thymidine incorporation was performed to investigate the cell proliferation in

Fig. 2. Histological analysis by Mayer’s ha¨ malaun and eosin staining. (A) In untreated femora (Con), morphology of the bony diaphyisis and cartilaginous epiphyisis appears normal. In (B) CryoDMSO and (C) Cryo femora, functional organization of the epiphyseal growth plate is lost and the periosteum appears disrupted and partially absent (3). Cellular necrosis in the bone marrow is generally less pronounced in CryoDMSO than in Cryo bones. Bar represents 100 ␮m and in the inserts 20 ␮m.

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Fig. 6. Characterization of B-cell and macrophage lineage cells by immunohistochemistry for CD45R/B220- and F4/80-antigens, respectively. In CryoDMSO the proliferative signal (3, black arrows) does not coincide with staining for (A) CD45R/B220- and (B) F4/80-antigens (3, red arrows). Bar represents 20 ␮m.

the organ culture. In Con (Fig. 3A) autoradiographic signals, visible as silver grains on the sections, were detectable on the surfaces of the cortical and cancellous bone, as well as at the edges of the cartilaginous epiphysis and in the proliferation zone of the epiphyseal growth plate. In the intramedullary space, only a few cells showed labeling with [methyl-3H]thymidine. In contrast to Con, where only occasional labeling with [methyl-3H]thymidine was observed in the intramedullary space, thymidine incorporation was strictly restricted to the marrow space in CryoDMSO (Fig. 3B). No thymidine incorporation into the nuclei of cells on

Fig. 5. Staining of osteoclasts by enzyme histochemistry for TRAP: (A) in Con femora, osteoclasts are attached to the cortical and cancellous bone surface (3, black arrow). TRAP-positive cells in (B) CryoDMSO and in (C) Cryo are mostly in close contact with the bone surface (3, black arrows), but part of the TRAP-positive cells are released from the bone and are located within the marrow cavity (3, green arrows). In all frozen femora cortical and trabecular surfaces are covered to a large extent with a thin layer which can not be assigned to specific cellular structures and which is positive for TRAP (3, red arrows). Bar represents 100 ␮m.

Fig. 3. Determination of cell proliferation by [methyl-3H]thymidine incorporation. Organ cultures were incubated for 24 h in the presence of [methyl-3H]thymidine. (A) In Con femora, autoradiogaphy reveals incorporation of the label in cells (3) in contact with bone and in cells at the edges of the cartilaginous epiphysis and in the proliferation zone of the epiphyseal growth plate. (B) No proliferation signals are observed in bone and cartilage cells of femora frozen in the presence of DMSO. Proliferation signals, however, are detected in cells of the marrow cavity (3). (C) No proliferation signals are visible in the entire femur when the tissue was frozen in the absence of DMSO. Bar represents 100 ␮m.

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bone and in cartilage was observed. Femora of Cryo (Fig. 3C) revealed no proliferation signals. In situ hybridization In situ hybridization for mRNA encoding col ␣1(I) was performed to detect active osteoblasts. In Con (Fig. 4A), osteoblasts synthesizing collagen were observed at the surface of the cortical and cancellous bone. mRNA encoding col ␣1(I) was further detected in the hypertrophic chondrocytes of the epiphyseal growth plate and at the edge of the epiphysis. In CryoDMSO (Fig. 4B) as well as in Cryo (Fig. 4C) no expression of mRNA encoding col ␣1(I) was observed. Enzyme histochemistry for TRAP Enzyme histochemistry for TRAP was performed to stain osteoclasts. In Con (Fig. 5A), osteoclasts were attached to the cortical bone surface and only a few were located at the cancellous bone surface. In cryopreserved femora (CryoDMSO, Cryo) (Fig. 5B and C) osteoclasts were mostly in close contact with the bone surface, but part of the TRAP-positive cells were released from the bone and located within the marrow cavity. In contrast to Con, in fast frozen femora cortical and trabecular surfaces were covered to a large extent with a thin layer which cannot be assigned to specific cellular structures and which is positive for TRAP. Immunohistochemistry for CD45R- and F4/80-antigens B-cell and macrophage lineage cells were investigated by immunohistochemistry for CD45R/B220- and F4/80antigens, respectively. Both markers revealed signals in the marrow space of all investigated femora. In CryoDMSO no [methyl-3H]thymidine uptake was detected in CD45R/ B220- and F4/80-positive cells (Fig. 6A and B). The distribution of the autoradiographic signals in this combined assay, only performed on CryoDMSO femora, was comparable to autoradiography done alone.

Discussion The present study was based on the hypothesis that the clinical success of osteochondral allograft transplantation might be improved by reducing antigenicity while simultaneously maintaining graft viability. This notion is supported by experimental evidence demonstrating that fast cryopreservation of allogeneic pancreatic islets in the presence

of DMSO improved the survival and the biological function [18]. It was suggested that the applied protocol maintained the viability of insulin-producing cells, while dendritic cells, macrophages, and lymphocytes were inactivated due to the sensitivity of these cells to fast freezing [19]. The purpose of this study was to investigate the biological function of osteochondral tissue in vitro after fast cryopreservation. Mature tissue would closer reflect the clinical situation of bone transplantation where mostly adult patients receive bone grafts of adult donors. However, neonatal osteochondral tissue is more active with respect to cell proliferation and matrix synthesis and has, furthermore, the advantage of assessing biological effects, as revascularization and osteogenesis, in in vivo models [24 –26]. Untreated control femora showed well-maintained morphology and biological function after 24 h in culture. The finding that chondrocytes synthesize col ␣1(I) mRNA reflects a dedifferentiation phenomenon, whereby the collagen synthesis of chondrocytes switches in cell culture from type II to type I collagen [34]. After rapid freezing no marked changes in the gross morphology of bone and cartilage were detected; the periosteum, however, was disrupted. Previous investigations on cryopreserved periosteum, harvested from the rabbit tibia, revealed optimal in vitro and in vivo function [35,36] when freezing was performed at a rate of ⫺1°C/min in the presence of DMSO. In contrast, no significant differences in tissue properties were observed when tissues frozen at ⫺10°C/min were compared to periosteum devitalized by repeated cycles of freezing– thawing, suggesting that accelerated freezing rates destroy the biological function of this tissue. A similar observation was made in the present study, as tissues frozen at ⫺70°C/ min showed greatly reduced biological functions with respect to cell proliferation or protein synthesis when compared to control tissues. It has been described before that slow and/or noncontrolled cryopreservation of bone maintained the proliferation of osteoblasts in vitro [22] and the angiogenic potential in vivo [25], emphasizing the protective effect of protocols including slow freezing rates on the biology of the tissues. The effects of controlled fast freezing protocols on bone and osteochondral tissues and on the function of bone cells, however, are not known. In untreated control femora after 24 h in culture, TRAP staining revealed osteoclasts to be present on the surface of the bone. Upon application of the rapid freezing protocols, independent of the presence or absence of DMSO, many TRAP-positive cells were released from the bone surface and located in the marrow space. Furthermore, cortical and trabecular surfaces were covered to a large extent with a thin layer which cannot be assigned to specific cellular structures and which is positive for TRAP. Together, these

Fig. 4. Detection of active osteoblasts by in situ hybridization for mRNA encoding col ␣1(I). (A) After a culture period of 24 h, col ␣1(I) mRNA is detected in Con femora in osteoblasts as well as in hypertrophic chondrocytes of the epiphyseal growth plate and at the edge of the epiphysis (3). No expression of mRNA encoding col ␣1(I) is observed in (B) CryoDMSO and in (C) Cryo femora. Bar represents 100 ␮m.

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histological observations reflect the inactivation and/or destruction of osteoclasts. The effects are similar to those observed after the exposure of osteoclasts to calcitonin, which blocks osteoclast activity and induces the detachment of the cells from the bone surface [37]. Moreover, the destruction of osteoclasts, which have been assigned to the mononuclear phagocytic system, may suggest that the cells of this lineage are particularly sensitive to fast cryopreservation. This will lead to a selective depletion of the main source of antigen-presenting cells in the frozen graft, accompanied with a decrease in its immunogenicity. In control femora, after 24 h in culture, a great number of bone and cartilage cells were in a proliferative state. In femora subjected to one of our freezing protocols, bone and cartilage cells did not proliferate. Proliferating cells, however, were detected in the marrow cavity after 24 h in culture. The presence of these cells was dependent on DMSO during the freezing protocol as no cell proliferation was observed when freezing was performed without the cryoprotectant. Whether DMSO exerted a direct effect on cell proliferation has not been investigated in this study. However, it is known that DMSO exerts an inhibitory effect on cell proliferation [38,39]. Therefore, our findings suggest that the freezing protocol in combination with DMSO, rather than DMSO alone, led to the observed phenomen. As yet, the nature of the proliferating cells could not be demonstrated. They are not part, however, of the B-cell and macrophage/monocyte lineages, since staining for CD45R/ B220- and F4/80-antigens did not coincide with the proliferative signal. Our group demonstrated previously that isogeneic or allogeneic transplanted rapidly frozen grafts showed accelerated revascularization only when frozen in the presence of DMSO [26]. Therefore, it is possible that the proliferating cells observed in CryoDMSO are involved in this process of revescularization, a prerequisite for successful graft incorporation [8]. To our knowledge, the effects exerted by fast, computercontrolled freezing in the presence of DMSO on the biology of osteochondral grafts have not been described. In the present study, we report that fast freezing blocked the biological function of osteoblasts, chondrocytes, periosteum, and osteoclasts. If DMSO was present during the cryopreservation, some residual cell proliferation and thus partial viability could be preserved within the marrow cavity of the osteochondral tissue. Both effects, the inactivation of immune cells [19] and the maintenance of cell viability, may be of importance in the revascularization of transplanted cryopreserved osteochondral grafts, as has been demonstrated recently by our group [26].

Acknowledgments This study was supported by grants from SNF (32-050771.97), AO/ASIF, and Sandoz-Foundation to Michael Leunig. Axel Sckell was a recipient of a Feodor

Lynen-Fellowship from the Alexander von HumboldtFoundation. Christian R. Fraitzl was supported by a Fellowship of the Maurice E. Mu¨ ller Foundation, Berne.

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