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Transplantation of marrow-derived mesenchymal stem cells and platelet-rich plasma during distraction osteogenesis—a preliminary result of three cases
Bone 35 (2004) 892 – 898 www.elsevier.com/locate/bone
Transplantation of marrow-derived mesenchymal stem cells and platelet-rich plasma during distraction osteogenesis—a preliminary result of three cases Hiroshi Kitoh*, Takahiko Kitakoji, Hiroki Tsuchiya, Hirohito Mitsuyama, Hiroshi Nakamura, Mitsuyasu Katoh, Naoki Ishiguro Department of Orthopaedic Surgery, Nagoya University School of Medicine, Showa-ku, Nagoya, Aichi 466-8550, Japan Received 31 January 2004; revised 28 May 2004; accepted 18 June 2004 Available online 28 July 2004
Abstract Clinical results of distraction osteogenesis with transplantation of marrow-derived mesenchymal stem cells (MSCs) and platelet-rich plasma (PRP) were reviewed in three femora and two tibiae of the two patients with achondroplasia and one patient with congenital pseudarthrosis of the tibia. MSCs derived from the iliac crest were cultured with osteogenic supplements and differentiated into osteoblastlike cells. PRP, which is known to contain several growth factors and coagulate immediately by a minute introduction of thrombin and calcium, was prepared just before transplantation. Culture-expanded osteoblast-like cells and autologous PRP were injected into the distracted callus with the thrombin–calcium mixture so that the PRP gel might develop within the injected site. Transplantation of MSCs and PRP was done at the lengthening and consolidation period in each patient. The target lengths were obtained in every leg without major complications and the average healing index was 23.0 days/cm (18.8–26.9 days/cm). Although these results are still preliminary, transplantation of osteoblast-like cells and PRP, which seemed to be a safe and minimally invasive cell therapy, could shorten the treatment period by acceleration of bone regeneration during distraction osteogenesis. D 2004 Elsevier Inc. All rights reserved. Keywords: Distraction osteogenesis; Mesenchymal stem cells; Platelet-rich plasma; Cell cultures
Introduction Distraction osteogenesis has been successfully used for limb lengthening in patients with limb length discrepancy, severe short stature due to skeletal dysplasias, and large bone defect that develop as a result of etiological reasons such as tumor, infection, and trauma. Although this treatment method is biological and has many advantages for these pathological conditions, the periods of external fixation and bone maturation is long which results in higher * Corresponding author. Department of Orthopaedic Surgery, Nagoya University School of Medicine, 65 Tsurumai-Cho, Showa-ku, Nagoya, Aichi 466-8550, Japan. Fax: +81 52 744 2260. E-mail address: [email protected] (H. Kitoh). 8756-3282/$ - see front matter D 2004 Elsevier Inc. All rights reserved. doi:10.1016/j.bone.2004.06.013
rates of complications such as pin track infection, joint contractures, pin loosening, delayed consolidations and fractures [1]. Decreasing the treatment period by accelerating bone formation of the distracted callus could reduce these complications. Bone marrow contains a population of multipotent mesenchymal stem cells (MSCs) that generate the progenitors for osteogenic, chondrogenic, adipocytic, and myogenic cells [2]. MSCs can be directed towards the osteogenic lineage in vitro if cultured in the presence of dexamethasone, h-glycerophosphate and ascorbic acid. Furthermore, MSCs are appropriate for donor tissue and may be used in clinical applications because they are easily isolated from a small aspirate of bone marrow and readily generate various cell types. Using the rat limb lengthening
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model, we have previously demonstrated that transplantation of marrow derived osteoblast-like cells with collagen gel into the distracted callus promoted new bone formation and shortened the consolidation period [3]. Biodegradable carriers or scaffolds are needed for MSCs transplantation and tissue growth. Heterologous or allogeneic materials are not appropriate for clinical feasibility because it carries potential risks of infection, immune responses and pathogen transmission. Platelet-rich plasma (PRP), which is developed from autologous blood with a cell separator, is rich in platelets and contains several growth factors including platelet-derived growth factor (PDGF), insulin-like growth factor (IGF-1), and transforming growth factors (TGF-h1 and h2) [4,5]. These growth factors enhance and accelerate the normal bone regeneration pathways [6]. Autologous PRP could be a suitable carrier for MSCs transplantation because it coagulates immediately by a minute introduction of calcium and thrombin. In this paper, we introduce a new cell therapy during distraction osteogenesis using culture expanded MSCs and autologous PRP. Preliminary results of the limb lengthening procedures with MSCs and PRP were reviewed.
Patients and methods Patients After informed consent was obtained from all individuals before surgery, 10 limb lengthening procedures with transplantation of MSCs and PRP were performed in seven patients at the Nagoya University between July 2002 and August 2003. Four femoral and two tibial lengthenings were performed in three achondroplasia patients bilaterally, and three femoral and one tibial lengthenings were done in four patients because of a limb length discrepancy, which was secondary to trauma (two limbs), congenital pseudarthrosis of the tibia (one), and developmental coxa vara (one). Of the seven patients, two patients with achondroplasia and one patient with congenital pseudarthrosis of the tibia were followed up until removal of the pins, while the remaining four patients were still under treatment. Surgical procedures Before the osteotomy, the Orthofix monolateral fixator was placed on the lateral aspect of the femur or on the anterolateral aspect of the tibia. Percutaneous osteotomy was performed in the diaphyseal region in each bone. Two to 3 cm at the junction of the distal and middle thirds of the fibular diaphysis was resected in conjunction with tibial lengthenings, and the distal part of the fibula was stabilized with a cannulated screw placed from the lateral malleolus to prevent proximal migration.
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Cell culture During the lengthening procedure, approximately 40 ml of bone marrow aspirates were collected from the iliac crest and harvested with sodium heparin and Dulbecco’s modified Eagle’s medium (DMEM; Sigma, St. Louis, MO, USA). Mononuclear cell fractions were isolated by centrifugation at 900 g for 20 min and plated at 5 106 cells/ cm2 into 75 cm2 culture flasks (Corning, NY, USA) in DMEM supplemented with penicillin–streptomycin (GibcoBRL, Life Technologies, Grand Island, NY, USA), 10% patients’ serum, 10-7 mol/l dexamethasone (Sigma), 10 mmol/lh-glycerophosphate (Sigma), and 50 Ag/ml ascorbic acid phosphate (Sigma). Nonadherent cells were removed from culture after 3 days by a series of phosphate-buffered saline (PBS) washes and subsequent medium changes. Adherent cells were expanded as monolayer cultures in a 5% CO2/95% air atmosphere at 378C with medium changes every 3 days. When cultured dishes became near confluent, cells were dissociated with 0.025% trypsin-EDTA (GibcoBRL, Life Technologies) and suspended at a density of 5000 cells/cm2 for continued passages. Half of the confluent firstpassaged cells were dissolved in preservation media and stored in liquid nitrogen for the second transplantation. MSCs cultured for nearly 3 weeks were applied for transplantation. Before transplantation, culture media were examined for contaminations of bacterium, fungus, mycoplasma, and pathological viruses such as hepatitis B, hepatitis C, and cytomegalovirus. For evaluation of osteoblastic differentiation of MSCs, the concentration of bone specific alkaline phosphatase (BAP) and carboxy-terminal propeptide of type I collagen (PICP) in the culture medium was measured in sequential saturation using commercially available enzyme immunoassay (EIA) and radioimmunoassay (RIA) respectively (SRL Inc, Japan). Each sample was measured in duplicates. MSCs treated without additional supplements were used as controls. Preparation of autologous PRP Approximately 200 ml of venous blood was drawn into a sterile bag containing sodium citrate as an anticoagulant for processing PRP. During the first centrifugation at 200 g for 15 min in a Refrigerated Centrifuge 9800 (Kubota Corporation, JAPAN), the components of blood were separated into two phases: one supernatant that constitutes PRP and one supernatant of erythrocytes and leukocytes. The samples of PRP underwent to a second centrifugation at 560 g for 15 min that allows the precipitation of the platelets. Total amount of PRP was finally concentrated according to the number of injection sites (5 ml of PRP per injection) by removing the superficial platelet poor plasma. Collecting venous blood and preparation of autologous PRP was commenced within 48 h before transplantation for fear of a decline in platelet function and increased risk of serious complication from bacterial contamination. Processed PRP
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was then stored with agitating at room temperature until transplantation. A sample of PRP was also used for determination of platelet count after processing. Transplantation of MSCs At the operating room, culture expanded MSCs were dissolved in a PRP, and 5000 units of human thrombin in the powder was mixed with 2 meq of calcium gluconate aseptically. Each mixture was prepared for the specific application in our cell therapy. Under X-ray guidance, two 18-gauge disposable spinal needles were inserted at the center of the distracted callus face to face with each tip (Fig. 1). Two milliliters of a thrombin–calcium mixture and 5 ml of a mixture of PRP and osteoblast-like cells were injected simultaneously into the callus so that the PRP gel might develop within the injected site.
Results Characterization of MSCs After several days in culture, a low number of fibroblastlike appearance MSCs was obtained, although the initial adherence of MSCs was diverse in each case. On approximately day 10, MSCs grew to reach a semiconfluent monolayer in primary culture with the average of 1.2 106 cells. The suspended cells reached confluence in 3–5 days and the third-passaged (P3) cells with the average of 2.3 107 (1.4 107–5.0 107) were used for transplantation.
Fig. 2. In vitro osteogenic differentiation of culture expanded MSCs from seven patients with passaging. BAP activity (A) and PICP concentration (B) in the culture medium. The columns show the means F SD.
Under the culture conditions with osteogenic supplements, morphological transformation of MSCs from an elongated to a more cuboidal shape was observed. Culture expanded MSCs were characterized for BAP activity and PICP secretion. The BAP activity within the culture medium was the highest in the P1 cells and maintained at all passages (Fig. 2A). On the other hand, a gradual increase was observed in the secretion of PICP during passaging (Fig. 2B). Platelet concentration of PRP PRP was prepared 14 times from seven patients (twice a patient) for transplantation. The average amount of 182 ml (158–200 ml) whole blood was drawn at a time. The average amount of PRP for transplantation was 9.9 ml (5–20 ml) after processing. The serum platelet concentration was averaged of 2.43 105/Al (1.53 105–3.2 105/Al) in a whole blood, while platelets reached an average concentration of 17.2 105/Al (14.0 105–19.8 105/Al) in a processed PRP, which was seven times the serum level. The specific growth factors in PRP were not quantified in this study. Case report
Fig. 1. The fluoroscopic image at the time of transplantation. The MSCs– PRP mixture and the thrombin–calcium mixture were simultaneously injected into the distracted callus through the 18-gauge disposable spinal needles.
Four patients who were still under treatment were not included for clinical assessment because total duration of treatment (from the surgery to removal of the pins) could not
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Fig. 3. Radiographs of the right femur of Case 1 before the surgery (A), and just after removal of the pins (B).
be determined in these cases. The remaining three patients (three femoral and two tibial lengthenings) who finished removal of the pins were evaluated for clinical feasibility of the treatment. Case 1 is a 15-year-old female affected with achondroplasia and bilateral femoral lengthenings were performed. Distraction at 0.5 mm twice a day (1 mm/day) was begun 7
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days after the operation. On the 21st day, 1.7 107 culture expanded MSCs were available and injected at one site in each femur. The distraction was increased to 1.5 mm/day for 2 weeks (34th–47th day) because callus formation was likely to consolidate prematurely. The femora were lengthened 10 cm over a period of 96 days. At the end of the lengthening phase, the second percutaneous injection of 2.1 107 MSCs was done into two sites of the regenerate in each femur. The consolidation period was 127 days and the total duration of treatment was 230 days (Figs. 3A and B). The healing indices were 23.0 days/cm on both femora. Case 2 is a 14-year-old female of right congenital pseudarthrosis of the tibia. Although bony union of the pseudarthrosis lesion was achieved by the vascularized fibular graft performed at the age of 5 years, limb length discrepancy due to shortness of the right tibia remained for residual deformity. For fear of a poor osteogenic ability of her right pathological tibia, femoral lengthening instead of tibial lengthening was done for correction. Distraction was begun at a rate of 1.0 mm/day after a waiting period of 14 days. Culture expanded MSCs of 1.8 107 cells were injected into the distracted callus on the 23rd day after the surgery under local anesthesia (Figs. 4A and B). Against premature consolidation, the distraction rate was increased to 1.5 mm/day for 1 week (35th–41st day). Distraction was ceased when no limb length discrepancy was confirmed radiographically. Total amount of lengthening was 3.6 cm over a period of 33 days. After a 20-day period of consolidation since the end of distraction, 1.4 107 osteoblast-like cells were percutaneously transplanted within the regenerate. The consolidation period was 50 days and the entire duration of the external fixation was 97 days. The healing index was 26.9 days/cm. Case 3 is a 13-year-old achondroplastic male and bilateral tibial lengthenings were performed. After an initial delay of 10 days, gradual distraction at a rate of 1.0 mm/day
Fig. 4. Radiographs of the right femur of Case 2 before the first transplantation of MSCs (A), and 2 weeks later after transplantation (B). Callus formation was enhanced within the distracted area.
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Fig. 5. Radiographs of the left lower limb of Case 3 before the first transplantation of MSCs (A), 2 weeks later after the first transplantation (B), and at the end of lengthening (just before the second transplantation) (C). Callus formation was satisfactory during the lengthening period.
was commenced. Twenty-one days after the operation, 3.2 107 MSCs were subcutaneously injected at one site on each tibia under local anesthesia (Figs. 5A and B). The distraction rate was increased to 1.5 mm/day for 2 weeks (35th–48th day) so that it prevented an early consolidation. Total of 10 cm lengthening was gained over a period of 96 days (Fig. 5C). MSCs (2.8 107) were administered in two sites on each tibia at the end of the lengthening. The consolidation phase was 83 days in the left tibia and 128 days in the right tibia. The healing index was 18.8 and 23.3 days/cm, respectively. Clinical assessment of the treatment We examined eight achondroplasia patients who underwent 14 femoral and 16 tibial lengthenings without MSCs transplantation at our institution using the Orthofix monolateral fixator. These patients (controls) were compared to two achondroplasia patients treated with MSCs and PRP transplantation (Case 1 and Case 3) for clinical assessment of the treatment. The average age at the surgery and the amount of lengthening were 16.7 years and 8.7 cm in controls, and 13.9 years and 10 cm in our series, respectively. The healing index of control patients ranged from 22.3 to 65.7 days/cm with an average of 37.8 days/cm. On the other hand, the average healing index of Case 1 and Case 3 was 22.0 days/cm (range, 18.8 to 23.3 days/cm).
Discussion The incidence and severity of complications during limb lengthening tends to increase in relation to the period of external fixation. Many attempts using electronic stimula-
tion, hyperbaric oxygen exposure, low intensity pulsed ultrasound stimulation, or systemic administration of recombinant growth hormone, have been made to promote bone formation during consolidation period [7–10]. Enhanced new bone regenerates have been demonstrated in rabbit models of distraction osteogenesis by transplantation of fresh bone marrow cells, cultured periosteal cells, or recombinant human bone morphogenetic protein-2 (rhBMP-2) loaded onto an absorbable collagen sponge [11– 13]. Tissue engineering has emerged as a possible alternative strategy to accelerate bone formation and shorten the treatment period. Three components are essential for this strategy: osteogenic cells, osteoinductive factors, and a scaffold that mimics the structural environment to promote bone regeneration. Several authors have reported that human MSCs preserved the phenotype of osteoblastic lineage and potency of differentiation into active osteoblastic cells in response to osteogenic supplements including dexamethasone [14–16]. In the culture system with osteogenic supplements, we have previously reported that the concentration of osteogenic markers showed a peak at 12 days after the first subculture [3]. Considering additional 10 days for primary culture, expanded MSCs at the 21st–23rd day were applied for transplantation in the present study. The number of cells for transplantation, on the other hand, was not standardized and depended on the number of available MSCs after ex vivo expansion. With regard to osteoblastic differentiation of culture expanded MSCs, the activity of BAP and PICP was increased, but the amounts of other osteogenic markers including osteocalcin, which is a marker of matured osteoblasts, were not examined in this study. Further study is necessary to determine the optimal number and osteoblastic phenotype of MSCs for transplantation.
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PRP is derived from an autogenous reparation of concentrated platelets and contains many growth factors including PDGF, TGF-h1 and h2, and IGF-1. PDGF seems to have numerous positive effects on wound healing including mitogenesis, angiogenesis, and upregulation of other growth factors [17]. TGF-hs are general growth and differentiating factors involved with bone regeneration in mitogenesis of osteoblast precursors [18]. Additionally, they inhibit osteoclast formation and bone resorption. IGF-1 is thought to increase numbers of osteoblasts and thereby accelerate bone formation [19]. Higher platelet concentration in PRP than in whole blood (approximately sevenfold) confirmed successful processing of PRP, which would contain multiple growth factors within the platelets, although the amounts of these factors were not quantified. Since these platelet-derived growth factors are released and activated during degranulation of the alpha granules [20], the potential loss of bioactivity of the growth factors may be concerned during storage of PRP. Less than 48 h for storage would be allowable for the better retention of platelet properties [21,22], and a continuous gentle agitation of the platelet concentrates at room temperature would contribute to the maintenance of platelet viability [23]. Recently, PRP gel has been used successfully as a scaffold for bone formation [24,25] and it enhanced bone regeneration when used in conjunction with autologous bone graft in the area of reconstructive oral and maxillofacial surgery [4,26]. In the area of orthopedic surgery such as spinal fusions; however, the efficacy of PRP gel with bone graft is still controversial [27,28]. This is the first report, to our knowledge, delineating a useful application of PRP in distraction osteogenesis of the long bones. In our preliminary in vitro experiments, the PRP–MSCs solution immediately coagulated and formed gel by mixing the thrombin– calcium solution; thus, the mixture could hardly be injected through the needles. In this study, therefore, they were injected separately and allowed to form a gel within the injected sites for minimally invasive surgery. A waiting period of the patients was determined individually based on an expected early callus formation, which was better in achondroplasia rather than healthy children, and femoral osteotomy rather than tibial osteotomy in principle. On the other hand, the distraction rate of 1.0 mm/day is a standard in limb lengthenings, but it could be changed in relation to the status of the callus and tension within the soft tissues. Since enhanced callus formation was observed radiographically after the first transplantation of MSCs and PRP in all three cases, the distraction was accelerated for a short term with great care for soft tissue tension, resulting in a shortened lengthening period. Since the consolidation period is approximately twice to three times as long as the lengthening period, shortening of the lengthening period theoretically contributes to the reduction of total treatment period. In Case 3, in spite of aggressive stretching exercise of the Achilles tendon and antiequinovarus splint at night, more severe equinovarus deformity
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progressed in the right foot at the end of the lengthening phase. Differences in consolidation period between the both legs may be due to disproportionate compression stresses exerted by the weightbearing, which provides the beneficial effect on bone consolidation [29]. The results of distraction osteogenesis are currently assessed on the basis of the amount of length gained, the prevalence of associated complications or additional procedures, and quantitative parameters such as healing index [30]. In the present study, the planned lengthening could be achieved in all legs without additional procedures or complications except for superficial pin track infection. The healing index has been estimated as a quantitative parameter but it depends on several parameters such as the patient’s age, amount of length gained, and the location of the osteotomy (generally, it tends to decrease in children, with length gained, and in femoral lengthening rather than tibial lengthening) [31,32]. Therefore, comparison between different series of patients is difficult because of differences in diagnosis, lengthening techniques, and types of external fixation. In the current study, diagnosis (achodroplasia) and type of external fixation (Orthofix monolateral fixator) matched 30 limb lengthenings without MSCs transplantation were selected as controls, although there was great variability between patients. Remarkable reduction of the healing index was demonstrated in our series compared to matched controls. Indeed in Case 1, bilateral tibial lengthenings of 10 cm were performed by conventional method 1 year before the surgery and the healing index was 38.7 days/ cm. Although these are preliminary clinical results for limited cases, transplantation of marrow derived osteoblastlike cells with PRP could shortened the treatment period and lessen the associated complications by accelerating new bone formation during distraction osteogenesis. Autologous cell therapy for bone regeneration by combination of MSCs and PRP has many advantages in clinical feasibility. First, MSCs can be expanded ex vivo and manipulated into osteoblastic lineage without difficulty by culturing with osteogenic supplements including dexamethasone. Second, the treatment is safe with minimal side effect because both MSCs and PRP are autologous which are nontoxic and nonimmunoreactive. Third, transplantation of MSCs by injection is less invasive resulting in low risks of infection. The technique we proposed may be applicable for the repair of bone defects, and could be a useful alternative to allogeneic or autologous bone grafts, which appear to be safe, minimally invasive, and easy to perform, with great potential in clinical applications.
Acknowledgment This work was supported in part by a Grant-in-Aid for Scientific Research from the Ministry of Education, Culture, Sports, Science and Technology of Japan (contact grant number 14370461 and 15591573).
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References [17] [1] Simpson AH, Kenwright J. Fracture after distraction osteogenesis. J Bone Joint Surg 2000;82-B:659 – 65. [2] Pittenger MF, Mackay AM, Beck SC, Jaiswal RK, Douglas R, Mosca JD, et al. Multilineage potential of adult human mesenchymal stem cells. Science 1999;284:143 – 7. [3] Takamine Y, Tsuchiya H, Kitakoji T, Kurita K, Ono Y, Ohshima Y, et al. Distraction osteogenesis enhanced by osteoblastlike cells and collagen gel. Clin Orthop 2002;399:240 – 6. [4] Marx RE, Carlson ER, Eichstaedt RM, Schimmele SR, Strauss JE, Georgeff KR. Platelet-rich plasma: growth factor enhancement for bone grafts. Oral Surg Oral Med Oral Pathol Oral Radiol Endod 1998;85:638 – 46. [5] Schilephake H. Bone growth factors in maxillofacial skeletal reconstruction. Int J Oral Maxillofac Surg 2002;31:469 – 84. [6] Landesberg R, Roy M, Glickman RS. Quantification of growth factor levels using simplified method of platelet-rich plasma gel preparation. J Oral Maxillofac Surg 2000;58:297 – 300. [7] Pepper JR, Herbert MA, Anderson JR, Bobechko WP. Effect of capacitive coupled electrical stimulation on regenerate bone. J Orthop Res 1996;14:296 – 302. [8] Kitakoji T, Takashi S, Ono Y, Hattori T, Takahashi H, Iwata H. Effect of hyperbaric oxygenation treatment on lengthened callus. Undersea Hyperb Med 1999;26:165 – 8. [9] Sato W, Matsushima T, Nakamura K. Acceleration of increase in bone mineral content by low-intensity ultrasound energy in leg lengthening. J Ultrasound Med 1999;18:699 – 702. [10] Raschke MJ, Bail H, Windhagen HJ, Kolbeck SF, Weiler A, Raun K, et al. Recombinant growth hormone accelerates bone regenerate consolidation in distraction osteogenesis. Bone 1999;24:81 – 8. [11] Hamanishi C, Yoshii T, Totani Y, Tanaka S. Bone mineral density of lengthened rabbit tibia is enhanced by transplantation of fresh autologous bone marrow cells. Clin Orthop 1994;303:250 – 5. [12] Takushima A, Kitano Y, Harii K. Osteogenic potential of cultured periosteal cells in a distracted bone gap in rabbits. J Surg Res 1998;78:68 – 77. [13] Li G, Bouxsein ML, Luppen C, Li XJ, Wood M, Seeherman HJ, et al. Bone consolidation is enhanced by rhBMP-2 in a rabbit model of distraction osteogenesis. J Orthop Res 2002;20:779 – 88. [14] Cheng S, Yang JW, Rifas L, Zhang S, Avioli LV. Differentiation of human bone marrow osteogenic stromal cells in vitro: induction of the osteoblast phenotype by dexamethasone. Endocrinology 1994; 134:277 – 86. [15] Jaiswal N, Haynesworth SE, Caplan AI, Bruder SP. Osteogenic differentiation of purified, culture-expanded human mesenchymal stem cells in vitro. J Cell Biochem 1997;64:295 – 312. [16] Yamanouchi K, Satomura K, Gotoh Y, Kitaoka E, Tobiume S, Kume K, et al. Bone formation by transplanted human osteoblasts cultured
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Transplantation of marrow-derived mesenchymal stem cells and platelet-rich plasma during distraction osteogenesis—a preliminary result of three cases Hiroshi Kitoh*, Takahiko Kitakoji, Hiroki Tsuchiya, Hirohito Mitsuyama, Hiroshi Nakamura, Mitsuyasu Katoh, Naoki Ishiguro Department of Orthopaedic Surgery, Nagoya University School of Medicine, Showa-ku, Nagoya, Aichi 466-8550, Japan Received 31 January 2004; revised 28 May 2004; accepted 18 June 2004 Available online 28 July 2004
Abstract Clinical results of distraction osteogenesis with transplantation of marrow-derived mesenchymal stem cells (MSCs) and platelet-rich plasma (PRP) were reviewed in three femora and two tibiae of the two patients with achondroplasia and one patient with congenital pseudarthrosis of the tibia. MSCs derived from the iliac crest were cultured with osteogenic supplements and differentiated into osteoblastlike cells. PRP, which is known to contain several growth factors and coagulate immediately by a minute introduction of thrombin and calcium, was prepared just before transplantation. Culture-expanded osteoblast-like cells and autologous PRP were injected into the distracted callus with the thrombin–calcium mixture so that the PRP gel might develop within the injected site. Transplantation of MSCs and PRP was done at the lengthening and consolidation period in each patient. The target lengths were obtained in every leg without major complications and the average healing index was 23.0 days/cm (18.8–26.9 days/cm). Although these results are still preliminary, transplantation of osteoblast-like cells and PRP, which seemed to be a safe and minimally invasive cell therapy, could shorten the treatment period by acceleration of bone regeneration during distraction osteogenesis. D 2004 Elsevier Inc. All rights reserved. Keywords: Distraction osteogenesis; Mesenchymal stem cells; Platelet-rich plasma; Cell cultures
Introduction Distraction osteogenesis has been successfully used for limb lengthening in patients with limb length discrepancy, severe short stature due to skeletal dysplasias, and large bone defect that develop as a result of etiological reasons such as tumor, infection, and trauma. Although this treatment method is biological and has many advantages for these pathological conditions, the periods of external fixation and bone maturation is long which results in higher * Corresponding author. Department of Orthopaedic Surgery, Nagoya University School of Medicine, 65 Tsurumai-Cho, Showa-ku, Nagoya, Aichi 466-8550, Japan. Fax: +81 52 744 2260. E-mail address: [email protected] (H. Kitoh). 8756-3282/$ - see front matter D 2004 Elsevier Inc. All rights reserved. doi:10.1016/j.bone.2004.06.013
rates of complications such as pin track infection, joint contractures, pin loosening, delayed consolidations and fractures [1]. Decreasing the treatment period by accelerating bone formation of the distracted callus could reduce these complications. Bone marrow contains a population of multipotent mesenchymal stem cells (MSCs) that generate the progenitors for osteogenic, chondrogenic, adipocytic, and myogenic cells [2]. MSCs can be directed towards the osteogenic lineage in vitro if cultured in the presence of dexamethasone, h-glycerophosphate and ascorbic acid. Furthermore, MSCs are appropriate for donor tissue and may be used in clinical applications because they are easily isolated from a small aspirate of bone marrow and readily generate various cell types. Using the rat limb lengthening
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model, we have previously demonstrated that transplantation of marrow derived osteoblast-like cells with collagen gel into the distracted callus promoted new bone formation and shortened the consolidation period [3]. Biodegradable carriers or scaffolds are needed for MSCs transplantation and tissue growth. Heterologous or allogeneic materials are not appropriate for clinical feasibility because it carries potential risks of infection, immune responses and pathogen transmission. Platelet-rich plasma (PRP), which is developed from autologous blood with a cell separator, is rich in platelets and contains several growth factors including platelet-derived growth factor (PDGF), insulin-like growth factor (IGF-1), and transforming growth factors (TGF-h1 and h2) [4,5]. These growth factors enhance and accelerate the normal bone regeneration pathways [6]. Autologous PRP could be a suitable carrier for MSCs transplantation because it coagulates immediately by a minute introduction of calcium and thrombin. In this paper, we introduce a new cell therapy during distraction osteogenesis using culture expanded MSCs and autologous PRP. Preliminary results of the limb lengthening procedures with MSCs and PRP were reviewed.
Patients and methods Patients After informed consent was obtained from all individuals before surgery, 10 limb lengthening procedures with transplantation of MSCs and PRP were performed in seven patients at the Nagoya University between July 2002 and August 2003. Four femoral and two tibial lengthenings were performed in three achondroplasia patients bilaterally, and three femoral and one tibial lengthenings were done in four patients because of a limb length discrepancy, which was secondary to trauma (two limbs), congenital pseudarthrosis of the tibia (one), and developmental coxa vara (one). Of the seven patients, two patients with achondroplasia and one patient with congenital pseudarthrosis of the tibia were followed up until removal of the pins, while the remaining four patients were still under treatment. Surgical procedures Before the osteotomy, the Orthofix monolateral fixator was placed on the lateral aspect of the femur or on the anterolateral aspect of the tibia. Percutaneous osteotomy was performed in the diaphyseal region in each bone. Two to 3 cm at the junction of the distal and middle thirds of the fibular diaphysis was resected in conjunction with tibial lengthenings, and the distal part of the fibula was stabilized with a cannulated screw placed from the lateral malleolus to prevent proximal migration.
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Cell culture During the lengthening procedure, approximately 40 ml of bone marrow aspirates were collected from the iliac crest and harvested with sodium heparin and Dulbecco’s modified Eagle’s medium (DMEM; Sigma, St. Louis, MO, USA). Mononuclear cell fractions were isolated by centrifugation at 900 g for 20 min and plated at 5 106 cells/ cm2 into 75 cm2 culture flasks (Corning, NY, USA) in DMEM supplemented with penicillin–streptomycin (GibcoBRL, Life Technologies, Grand Island, NY, USA), 10% patients’ serum, 10-7 mol/l dexamethasone (Sigma), 10 mmol/lh-glycerophosphate (Sigma), and 50 Ag/ml ascorbic acid phosphate (Sigma). Nonadherent cells were removed from culture after 3 days by a series of phosphate-buffered saline (PBS) washes and subsequent medium changes. Adherent cells were expanded as monolayer cultures in a 5% CO2/95% air atmosphere at 378C with medium changes every 3 days. When cultured dishes became near confluent, cells were dissociated with 0.025% trypsin-EDTA (GibcoBRL, Life Technologies) and suspended at a density of 5000 cells/cm2 for continued passages. Half of the confluent firstpassaged cells were dissolved in preservation media and stored in liquid nitrogen for the second transplantation. MSCs cultured for nearly 3 weeks were applied for transplantation. Before transplantation, culture media were examined for contaminations of bacterium, fungus, mycoplasma, and pathological viruses such as hepatitis B, hepatitis C, and cytomegalovirus. For evaluation of osteoblastic differentiation of MSCs, the concentration of bone specific alkaline phosphatase (BAP) and carboxy-terminal propeptide of type I collagen (PICP) in the culture medium was measured in sequential saturation using commercially available enzyme immunoassay (EIA) and radioimmunoassay (RIA) respectively (SRL Inc, Japan). Each sample was measured in duplicates. MSCs treated without additional supplements were used as controls. Preparation of autologous PRP Approximately 200 ml of venous blood was drawn into a sterile bag containing sodium citrate as an anticoagulant for processing PRP. During the first centrifugation at 200 g for 15 min in a Refrigerated Centrifuge 9800 (Kubota Corporation, JAPAN), the components of blood were separated into two phases: one supernatant that constitutes PRP and one supernatant of erythrocytes and leukocytes. The samples of PRP underwent to a second centrifugation at 560 g for 15 min that allows the precipitation of the platelets. Total amount of PRP was finally concentrated according to the number of injection sites (5 ml of PRP per injection) by removing the superficial platelet poor plasma. Collecting venous blood and preparation of autologous PRP was commenced within 48 h before transplantation for fear of a decline in platelet function and increased risk of serious complication from bacterial contamination. Processed PRP
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was then stored with agitating at room temperature until transplantation. A sample of PRP was also used for determination of platelet count after processing. Transplantation of MSCs At the operating room, culture expanded MSCs were dissolved in a PRP, and 5000 units of human thrombin in the powder was mixed with 2 meq of calcium gluconate aseptically. Each mixture was prepared for the specific application in our cell therapy. Under X-ray guidance, two 18-gauge disposable spinal needles were inserted at the center of the distracted callus face to face with each tip (Fig. 1). Two milliliters of a thrombin–calcium mixture and 5 ml of a mixture of PRP and osteoblast-like cells were injected simultaneously into the callus so that the PRP gel might develop within the injected site.
Results Characterization of MSCs After several days in culture, a low number of fibroblastlike appearance MSCs was obtained, although the initial adherence of MSCs was diverse in each case. On approximately day 10, MSCs grew to reach a semiconfluent monolayer in primary culture with the average of 1.2 106 cells. The suspended cells reached confluence in 3–5 days and the third-passaged (P3) cells with the average of 2.3 107 (1.4 107–5.0 107) were used for transplantation.
Fig. 2. In vitro osteogenic differentiation of culture expanded MSCs from seven patients with passaging. BAP activity (A) and PICP concentration (B) in the culture medium. The columns show the means F SD.
Under the culture conditions with osteogenic supplements, morphological transformation of MSCs from an elongated to a more cuboidal shape was observed. Culture expanded MSCs were characterized for BAP activity and PICP secretion. The BAP activity within the culture medium was the highest in the P1 cells and maintained at all passages (Fig. 2A). On the other hand, a gradual increase was observed in the secretion of PICP during passaging (Fig. 2B). Platelet concentration of PRP PRP was prepared 14 times from seven patients (twice a patient) for transplantation. The average amount of 182 ml (158–200 ml) whole blood was drawn at a time. The average amount of PRP for transplantation was 9.9 ml (5–20 ml) after processing. The serum platelet concentration was averaged of 2.43 105/Al (1.53 105–3.2 105/Al) in a whole blood, while platelets reached an average concentration of 17.2 105/Al (14.0 105–19.8 105/Al) in a processed PRP, which was seven times the serum level. The specific growth factors in PRP were not quantified in this study. Case report
Fig. 1. The fluoroscopic image at the time of transplantation. The MSCs– PRP mixture and the thrombin–calcium mixture were simultaneously injected into the distracted callus through the 18-gauge disposable spinal needles.
Four patients who were still under treatment were not included for clinical assessment because total duration of treatment (from the surgery to removal of the pins) could not
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Fig. 3. Radiographs of the right femur of Case 1 before the surgery (A), and just after removal of the pins (B).
be determined in these cases. The remaining three patients (three femoral and two tibial lengthenings) who finished removal of the pins were evaluated for clinical feasibility of the treatment. Case 1 is a 15-year-old female affected with achondroplasia and bilateral femoral lengthenings were performed. Distraction at 0.5 mm twice a day (1 mm/day) was begun 7
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days after the operation. On the 21st day, 1.7 107 culture expanded MSCs were available and injected at one site in each femur. The distraction was increased to 1.5 mm/day for 2 weeks (34th–47th day) because callus formation was likely to consolidate prematurely. The femora were lengthened 10 cm over a period of 96 days. At the end of the lengthening phase, the second percutaneous injection of 2.1 107 MSCs was done into two sites of the regenerate in each femur. The consolidation period was 127 days and the total duration of treatment was 230 days (Figs. 3A and B). The healing indices were 23.0 days/cm on both femora. Case 2 is a 14-year-old female of right congenital pseudarthrosis of the tibia. Although bony union of the pseudarthrosis lesion was achieved by the vascularized fibular graft performed at the age of 5 years, limb length discrepancy due to shortness of the right tibia remained for residual deformity. For fear of a poor osteogenic ability of her right pathological tibia, femoral lengthening instead of tibial lengthening was done for correction. Distraction was begun at a rate of 1.0 mm/day after a waiting period of 14 days. Culture expanded MSCs of 1.8 107 cells were injected into the distracted callus on the 23rd day after the surgery under local anesthesia (Figs. 4A and B). Against premature consolidation, the distraction rate was increased to 1.5 mm/day for 1 week (35th–41st day). Distraction was ceased when no limb length discrepancy was confirmed radiographically. Total amount of lengthening was 3.6 cm over a period of 33 days. After a 20-day period of consolidation since the end of distraction, 1.4 107 osteoblast-like cells were percutaneously transplanted within the regenerate. The consolidation period was 50 days and the entire duration of the external fixation was 97 days. The healing index was 26.9 days/cm. Case 3 is a 13-year-old achondroplastic male and bilateral tibial lengthenings were performed. After an initial delay of 10 days, gradual distraction at a rate of 1.0 mm/day
Fig. 4. Radiographs of the right femur of Case 2 before the first transplantation of MSCs (A), and 2 weeks later after transplantation (B). Callus formation was enhanced within the distracted area.
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Fig. 5. Radiographs of the left lower limb of Case 3 before the first transplantation of MSCs (A), 2 weeks later after the first transplantation (B), and at the end of lengthening (just before the second transplantation) (C). Callus formation was satisfactory during the lengthening period.
was commenced. Twenty-one days after the operation, 3.2 107 MSCs were subcutaneously injected at one site on each tibia under local anesthesia (Figs. 5A and B). The distraction rate was increased to 1.5 mm/day for 2 weeks (35th–48th day) so that it prevented an early consolidation. Total of 10 cm lengthening was gained over a period of 96 days (Fig. 5C). MSCs (2.8 107) were administered in two sites on each tibia at the end of the lengthening. The consolidation phase was 83 days in the left tibia and 128 days in the right tibia. The healing index was 18.8 and 23.3 days/cm, respectively. Clinical assessment of the treatment We examined eight achondroplasia patients who underwent 14 femoral and 16 tibial lengthenings without MSCs transplantation at our institution using the Orthofix monolateral fixator. These patients (controls) were compared to two achondroplasia patients treated with MSCs and PRP transplantation (Case 1 and Case 3) for clinical assessment of the treatment. The average age at the surgery and the amount of lengthening were 16.7 years and 8.7 cm in controls, and 13.9 years and 10 cm in our series, respectively. The healing index of control patients ranged from 22.3 to 65.7 days/cm with an average of 37.8 days/cm. On the other hand, the average healing index of Case 1 and Case 3 was 22.0 days/cm (range, 18.8 to 23.3 days/cm).
Discussion The incidence and severity of complications during limb lengthening tends to increase in relation to the period of external fixation. Many attempts using electronic stimula-
tion, hyperbaric oxygen exposure, low intensity pulsed ultrasound stimulation, or systemic administration of recombinant growth hormone, have been made to promote bone formation during consolidation period [7–10]. Enhanced new bone regenerates have been demonstrated in rabbit models of distraction osteogenesis by transplantation of fresh bone marrow cells, cultured periosteal cells, or recombinant human bone morphogenetic protein-2 (rhBMP-2) loaded onto an absorbable collagen sponge [11– 13]. Tissue engineering has emerged as a possible alternative strategy to accelerate bone formation and shorten the treatment period. Three components are essential for this strategy: osteogenic cells, osteoinductive factors, and a scaffold that mimics the structural environment to promote bone regeneration. Several authors have reported that human MSCs preserved the phenotype of osteoblastic lineage and potency of differentiation into active osteoblastic cells in response to osteogenic supplements including dexamethasone [14–16]. In the culture system with osteogenic supplements, we have previously reported that the concentration of osteogenic markers showed a peak at 12 days after the first subculture [3]. Considering additional 10 days for primary culture, expanded MSCs at the 21st–23rd day were applied for transplantation in the present study. The number of cells for transplantation, on the other hand, was not standardized and depended on the number of available MSCs after ex vivo expansion. With regard to osteoblastic differentiation of culture expanded MSCs, the activity of BAP and PICP was increased, but the amounts of other osteogenic markers including osteocalcin, which is a marker of matured osteoblasts, were not examined in this study. Further study is necessary to determine the optimal number and osteoblastic phenotype of MSCs for transplantation.
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PRP is derived from an autogenous reparation of concentrated platelets and contains many growth factors including PDGF, TGF-h1 and h2, and IGF-1. PDGF seems to have numerous positive effects on wound healing including mitogenesis, angiogenesis, and upregulation of other growth factors [17]. TGF-hs are general growth and differentiating factors involved with bone regeneration in mitogenesis of osteoblast precursors [18]. Additionally, they inhibit osteoclast formation and bone resorption. IGF-1 is thought to increase numbers of osteoblasts and thereby accelerate bone formation [19]. Higher platelet concentration in PRP than in whole blood (approximately sevenfold) confirmed successful processing of PRP, which would contain multiple growth factors within the platelets, although the amounts of these factors were not quantified. Since these platelet-derived growth factors are released and activated during degranulation of the alpha granules [20], the potential loss of bioactivity of the growth factors may be concerned during storage of PRP. Less than 48 h for storage would be allowable for the better retention of platelet properties [21,22], and a continuous gentle agitation of the platelet concentrates at room temperature would contribute to the maintenance of platelet viability [23]. Recently, PRP gel has been used successfully as a scaffold for bone formation [24,25] and it enhanced bone regeneration when used in conjunction with autologous bone graft in the area of reconstructive oral and maxillofacial surgery [4,26]. In the area of orthopedic surgery such as spinal fusions; however, the efficacy of PRP gel with bone graft is still controversial [27,28]. This is the first report, to our knowledge, delineating a useful application of PRP in distraction osteogenesis of the long bones. In our preliminary in vitro experiments, the PRP–MSCs solution immediately coagulated and formed gel by mixing the thrombin– calcium solution; thus, the mixture could hardly be injected through the needles. In this study, therefore, they were injected separately and allowed to form a gel within the injected sites for minimally invasive surgery. A waiting period of the patients was determined individually based on an expected early callus formation, which was better in achondroplasia rather than healthy children, and femoral osteotomy rather than tibial osteotomy in principle. On the other hand, the distraction rate of 1.0 mm/day is a standard in limb lengthenings, but it could be changed in relation to the status of the callus and tension within the soft tissues. Since enhanced callus formation was observed radiographically after the first transplantation of MSCs and PRP in all three cases, the distraction was accelerated for a short term with great care for soft tissue tension, resulting in a shortened lengthening period. Since the consolidation period is approximately twice to three times as long as the lengthening period, shortening of the lengthening period theoretically contributes to the reduction of total treatment period. In Case 3, in spite of aggressive stretching exercise of the Achilles tendon and antiequinovarus splint at night, more severe equinovarus deformity
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progressed in the right foot at the end of the lengthening phase. Differences in consolidation period between the both legs may be due to disproportionate compression stresses exerted by the weightbearing, which provides the beneficial effect on bone consolidation [29]. The results of distraction osteogenesis are currently assessed on the basis of the amount of length gained, the prevalence of associated complications or additional procedures, and quantitative parameters such as healing index [30]. In the present study, the planned lengthening could be achieved in all legs without additional procedures or complications except for superficial pin track infection. The healing index has been estimated as a quantitative parameter but it depends on several parameters such as the patient’s age, amount of length gained, and the location of the osteotomy (generally, it tends to decrease in children, with length gained, and in femoral lengthening rather than tibial lengthening) [31,32]. Therefore, comparison between different series of patients is difficult because of differences in diagnosis, lengthening techniques, and types of external fixation. In the current study, diagnosis (achodroplasia) and type of external fixation (Orthofix monolateral fixator) matched 30 limb lengthenings without MSCs transplantation were selected as controls, although there was great variability between patients. Remarkable reduction of the healing index was demonstrated in our series compared to matched controls. Indeed in Case 1, bilateral tibial lengthenings of 10 cm were performed by conventional method 1 year before the surgery and the healing index was 38.7 days/ cm. Although these are preliminary clinical results for limited cases, transplantation of marrow derived osteoblastlike cells with PRP could shortened the treatment period and lessen the associated complications by accelerating new bone formation during distraction osteogenesis. Autologous cell therapy for bone regeneration by combination of MSCs and PRP has many advantages in clinical feasibility. First, MSCs can be expanded ex vivo and manipulated into osteoblastic lineage without difficulty by culturing with osteogenic supplements including dexamethasone. Second, the treatment is safe with minimal side effect because both MSCs and PRP are autologous which are nontoxic and nonimmunoreactive. Third, transplantation of MSCs by injection is less invasive resulting in low risks of infection. The technique we proposed may be applicable for the repair of bone defects, and could be a useful alternative to allogeneic or autologous bone grafts, which appear to be safe, minimally invasive, and easy to perform, with great potential in clinical applications.
Acknowledgment This work was supported in part by a Grant-in-Aid for Scientific Research from the Ministry of Education, Culture, Sports, Science and Technology of Japan (contact grant number 14370461 and 15591573).
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