- Email: [email protected]
Demineralized Bone Matrix: Basic Science and Clinical Applications
Clin Podiatr Med Surg 22 (2005) 599 – 606
Demineralized Bone Matrix: Basic Science and Clinical Applications Douglas J. Pacaccio, DPM, Stephen F. Stern, DPMT Podiatric Surgery Section, Department of Orthopaedics, Inova Fairfax Hospital, 3300 Gallows Road, Falls Church, VA 22042, USA
Demineralized bone matrix (DBM) is one of many modalities that can be useful in foot and ankle surgery when used appropriately. Any surgeon who performs osseous work, whether it is fracture fixation or major reconstructive surgery, always faces the ultimate task of achieving bony union. Obtaining successful fusion of bone requires several factors be present and working in concert with one another. The multiple variables include local environment of the fusion site, systemic factors, physiologic factors, mechanical factors, and at times, fusion enhancers.
Basic science Despite relatively good results across most patient populations, there are times when a surgeon needs every advantage afforded to achieve the end goal of bony healing. Some examples include large defects in the desired healing site and patients who have osteoporosis, osteopenia, Charcot’s neuroarthropathy, or other conditions in which bony resorption occurs at a higher rate than bony deposition. Another patient population shown to be at a disadvantage for acute bone healing is smokers. Systemic nicotine is well known to retard osteogenesis and inhibit vascular ingrowth. Smoke extracts also cause calcitonin resistance, increase fracture end resorption, and decrease osteoblast function [1]. Other systemic factors include drugs like many chemotherapeutic agents and particularly nonsteroidal anti-inflammatory drugs. Several studies in the literature have shown that indo-
T Corresponding author. E-mail address: [email protected] (S.F. Stern). 0891-8422/05/$ – see front matter D 2005 Elsevier Inc. All rights reserved. doi:10.1016/j.cpm.2005.07.001 podiatric.theclinics.com
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methacin significantly inhibits spinal fusion compared with saline controls in rat models. The physiology of osteoporosis manifests changes in bone marrow quality and bone turnover. There are less osteogenic stem cells in the bone of elderly patients, which equals a decrease in the density of bone, further making the mechanical stability of fixation more difficult. Hormones can also play a role in adjusting the physiology of bone formation. Many growth hormones, by way of somatomedins, increase bone formation. Corticosteroids oppositely inhibit osteoblast differentiation and decrease matrix synthesis while weakening collagen bonds. Local factors include surface area of the fusion site, how well the ends are in apposition, and purposeful dissection preserving adequate blood supply. It is in these circumstances that various bone grafts and graft substitutes/enhancers are gaining favor in major reconstructive foot and ankle surgery along with standard procedures in higher risk patients. The first reported bone graft was canine skull xenograft used to repair a defect in a soldier’s skull. This graft was done in 1668 by Dutch surgeon Job Van Meekeren. On attempted removal of the graft, it was noted that the graft had completely healed the defect [2]. Grafts are often used to enhance either some or all of the physiologic and structural factors previously mentioned. Ideally, a graft has the same properties of healthy host bone, including the biology of bone repair coupled with mechanical stability. Depending on the type of graft, it can be considered an implant or simply a host-tissue transplant. Biodegradable grafts are preferred because they incorporate into the host bone and allow for remodeling as necessary for maximum functional outcomes. These grafts also eliminate stress risers produced by modulus gradients between host bone and permanent materials. There are three basic types of biodegradable bone grafts. Autograft is bone harvested from the recipient’s own body. Allograft is bone harvested from a donor of the same species. Xenograft is bone harvested from of donor of a different species, usually bovine or porcine. Xenograft is rarely, if ever, used today because of its antigenicity and the high rate of rejection seen in the past. A bone graft may have one, two, or all of the three basic properties of bone regeneration: osteogenicity, osteoinduction, and osteoconduction. Osteogenicity refers to the presence of viable osteoprogenitor cells within the graft. These cells aid in the early stages of bridging and uniting graft and host bone. Only autograft and bone marrow are osteogenic. Osteoinduction denotes the capability of stimulating pluripotential stem cells to differentiate into functioning osteogenic cells such as osteoblasts and osteoclasts. Allograft has osteoinductive and osteoconductive properties. Osteoconduction implies graft capacity to facilitate neovascularization and infiltration of the graft by osteogenic precursor cells by way of ‘‘creeping substitution.’’ Essentially, these types of grafts act only as scaffolding for new bone to bridge across. Examples of strictly osteoconductive grafts are calcium phosphate, ceramics, coral, and collagen. A graft’s net biologic contribution to any fusion site resides in it’s ability to display any or all of the three basic properties of bone regeneration, osteogenicity, osteoinduction, and osteoconduction. The ability to use inherent osteoblasts in
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early phases plus the capability to activate host osteoprogenitor cells coupled with facilitation of new bony ingrowth affect any graft’s viability. As the surgeon progresses the span from autograft to synthetic structural grafts, these biologic factors are decreased. The closer the graft is to having all three potentials, the less the graft relies on the host environment for replacement of graft tissue with host tissue. DBM is allogenic bone graft that has been processed into a putty or gel form. Although allogenic bone graft has been used by surgeons for the better part of 100 years, Barth [3] first proposed the concept of osteoinduction in 1893. Urist and McLean [4,5] further described its modern osteoinductive properties with relation to bone morphogenic proteins (BMP) in 1952. DBM is thought to possess more inductive properties than regular allograft because growth factor availability is increased after the demineralization process. DBM is different from cortical graft because it is mainly cancellous bone tissue. Cortical graft is desirable for structural support but has decreased osteogenicity relative to cancellous bone or marrow bone. Cortical bone has less surface area per unit weight and, therefore, less connectivity. The term connectivity describes the ability of an osteoconductive graft material to be ‘‘connected’’ to host bone and relates to the surface area available in the local healing environment for incorporation into the fusion mass. Because of the increased density of cortical bone, the rate of vascular ingrowth is slower. Although this construct presents increased relative mechanical strength at the time of surgery, it lags in mechanical strength and stability near the incorporation/remodeling phase of healing. The surgeon must weigh the delicate balance between the need for immediate structural support and the higher rate of incorporation that increased connectivity provides. Most often, regarding reconstructive forefoot and rearfoot surgery, a combination of cortical graft and DBM is preferred to maximize both structural support and connectivity of the fusion site. There are relatively scarce studies in the literature comparing DBM with autograft, synthetic graft, and other forms of allograft in foot and ankle surgery. Most of the literature on DBM and BMP is related to spine surgery because this is probably where these products are most commonly used. It was generally thought that autograft incorporates into host site faster than allograft, but Ragni and Lindholm [6] showed that DBM with hydroxyapatite (HA) demonstrated the quickest fusion rate compared with autograft and DBM or HA alone. These researchers used a rabbit interbody spine fusion model and ultimately concluded that at 6 months, the autograft results were comparable to the composite in fusion and mechanical strength. These findings may be the result of increased availability of BMP-saturated DBM (secondary to the demineralization process) potentiated by the increased connectivity of the graft site. There are several studies showing that DBM has a slightly higher concentration of BMP density than autograft. A study designed by Han and colleagues [7] showed that at 28 days, postintramuscular implantation of varying concentrations of DBM in an athymic nude rat model showed a direct correlation between DBM and mineralization of tissue. The implants were analyzed radiographically for mineral density. They were also analyzed for
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alkaline phosphatase activity on pluripotent myocytes. In both cases, the 100% active DBM displayed the most osteoinductive response, with each decrease in active DBM proportionally decreasing inductivity. These investigators described their results as having a ‘‘proportional osteoinductivity’’ response. Atti and colleagues [8] performed a similar study and reported the same conclusion. Yoo and coworkers [9] also showed that concentration of DBM improved osteoinductivity. They used DBM and bone marrow aspirate (BMA) in groups of skeletally mature rabbits. Two groups were implanted with varying amounts of DBM/HA/BMA, one group was implanted with DBM/HA only, and the two control groups were autograft only and BMA only. Of all the groups, it was found that those with the highest concentration of DBM, regardless of the BMA, showed the highest bone volumes in the fusion site. These investigators concluded that the amount of bone deposition was directly dependent on the amount of DBM present in the fusion site. Edwards [10] compared the osteoinductivity between a human DBM product with 100% DBM and one with 17% DBM in an athymic mouse population. Intramuscularly implanted DBM yielded ectopic bone samples that were sectioned at 14 days and 28 days. Histologic analysis revealed that 100% DBM outperformed 17% DBM in bone formation at both intervals. Graft healing occurs in five stages relatively similarly to simple fracture healing [11]. The first and second stages are marked by massive hemorrhage and inflammation, which occurs at the site of surgery as a result of surgical trauma. At these phases, the graft is infiltrated by polymorphonuclear leukocytes. During this transition, osteocytes die off but surface osteoblasts produce bone. Electronegativity gradients develop and create a physiologic environment conducive to bone healing. The gradient reduces pH and decreases oxygen tension. Relatively high oxygen tension signals mesenchymal stem cells to differentiate into osteoblasts and osteoclasts. Relatively low oxygen tension produces fibroblasts and chondroblasts from pleuripotential stem cells. Both scenarios occur in an environment with oxygen tension that is decreased from baseline. Aro and colleagues [12] showed that normal bone healing occurs in a relatively hypoxic environment. As the influx of inflammatory cells arrives at the fracture/ osteotomy site, the increased acidity decreases oxygen tension and stimulates osteoprogenitor stem cells. In a simple fracture, these osteoprogenitor stem cells are found mainly in the endosteum and cambium layer of periosteum. Marrow and host connective tissue also contains mesenchymal stem cells. It is at this phase that BMP-carrying DBM may aid in bony fusion by signaling stem cells, mainly through the transforming growth factor b superfamily, to become osteoprogenitor cells and begin the process of creating new bone. The relative early impact of BMP on the natural healing process possibly explains why autograft osteogenicity may have only little advantage in early healing over allograft. The advantage of increased concentration of BMP present in DBM, especially discernable in the previously cited studies, results in DBM composites achieving faster fusion. The third phase involves neovascularization of the graft. Host vessels invade the graft and provide nutrients for the following phase. This
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phase of healing can occur as soon as 2 days in cancellous (inductive) graft materials and slower in cortical graft. At the fourth phase, creeping substitution occurs as osteoblasts line the graft trebeculae and lay down osteoid. Creeping substitution is the term coined to describe the osteoclast cutting cones infiltrating necrotic bone followed by osteoblasts. At the fifth phase, the graft becomes remodeled from woven bone to lamellar bone, which has lower density of osteocytes and more parallel/organized collagen orientation. In addition, the medullary canal is created within the graft. Ideally, the host bone eventually replaces the graft and any entrapped graft is resorbed. Allograft bone is prepared for use by freezing, freeze-drying, or decalcifying. Simple freezing of allograft bone is known to be more antigenic than freezedrying or decalcifying. All forms of preparation destroy the viability of living osteocytes within the graft, thus removing any osteogenic properties. DBM is decalcified and maintains its inductive and conductive properties by preserving growth factors, BMPs, collagen, and matrix proteoglycans. The specimen is harvested no greater than 24 hours post mortem. Harvesting is done after performing a detailed medical history of the donor and donor’s family. The donor’s blood is tested for hepatitis A antibody, hepatitis B surface antigen, hepatitis B core antibody, hepatitis C antibody, HIV-1 virus antibodies, HIV direct antigen, human T-cell lymphoma virus-1 antibody, syphilis, and aerobic/anaerobic cultures. The surrounding tissues are also tested for surface and medullary contaminants (aerobic and anaerobic). The sample is irradiated with high-dose gamma radiation, and packaged and tested for sterility. Following initial testing, the specimen is stripped of any soft tissue and the bone is transported in bacitracin and polymyxin B solution. It is important to know that trace amounts of these antimicrobials remain in the final product, and patient hypersensitivity should be considered an absolute contraindication. The bone is milled into gross particle size when blood, marrow, and lipids are removed by washing with distilled water. The specimen is placed in an ultrasound bath before being cleared of lipids with 70% ethanol. The particles are flushed again with water and milled again to their final size. When particles reach their final size, they are demineralized in 0.6 M hydrochloric acid for 3 hours. The sample loses apatite crystals but retains its matrix calcium. The product is rinsed again with water and buffered with 0.1 M phosphate solution to the final pH. The sample undergoes two separate rounds of lyophilization (freeze-drying) to enable storage at room temperature. The sample is usually placed in its final packaging between rounds. Testing of room sterility, moisture, and calcium content finalizes the process. This process complies with the United States Pharmacopeia standards and the American Association of Tissue Banks [1,13]. Depending on the manufacturer, DBM comes in several forms. Grafton (Osteotech, Eatontown, New Jersey) was originally produced in a glycerol gel carrier. It is now available in putty form or as flexible sheets, which have been shown to produce higher fusion rates [14]. Osteofil (Regeneration Technologies, Alachua, Florida) is set in a hydrogel matrix. Opteform (Exactech, Gainesville, Florida) comes in a formable thermoplastic inert biodegradable carrier.
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Clinical applications Kado and colleagues [13] described various uses of Grafton DBM in the foot and ankle in 1996. Among the suggested applications are arthrodesis of the following joints: pantalar, ankle, triple (talonavicular, subtalar, and calcaneocuboid), Lisfranc’s, naviculocuneiform/intercuneiform, subtalar, and metatarsalphalangeal. They also performed osteotomies of the calcaneus (Silver, Evans) and midfoot (Cole). Other applications included tibiofibular fracture, metatarsal gunshot wound, Jones fracture nonunion, replaced donor site of autograft from calcaneus, medullary canal defect, first metatarsal cyst, and naviculocuneiform nonunion. These investigators reported good results and ease of application with the gel form of Grafton to fill gaps in bony fusion sites. Fig. 1 shows postoperative radiographs of a 72-year-old woman who underwent a triple arthrodesis and was found to have a fairly atrophic and soft navicular. She was noted to be osteopenic preoperatively, and several fixation modalities were requested to be present in the operating room as backup, including external fixation rails. Note that the first naviculocuneiform joint was also fused, which served to increase compression across the talonavicular joint and to plantarflex the medial column from elevatus to plantar grade. The doctor in residence, Dr. D. Pacaccio, and the attending physician assessed the medial column intraoperatively and agreed on the hypermobility present. It was believed that this fusion achieved adequate stability without locking up all the motion in the medial column by leaving the first metatarsal cuneiform joint intact. Washers
Fig. 1. (A,B) Triple arthrodesis with medial column stabilization in grossly unstable flat-foot deformity. Tricortical subtalar joint allograft with minimal gapping secondary to DBM implantation.
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were used with several of the screws because the bone was relatively soft. Femoral neck allograft was placed in the subtalar joint fusion site and the calcaneocuboid joint. It was decided intraoperatively to use Grafton DBM in the subtalar, calcaneocuboid, and talonavicular fusion sites and in the sinus tarsi. This application of DBM is a good one, in that it should help to create a bony bridge, adding to the stability of the fusion. The patient had an extremely planovalgus foot preoperatively and has a fairly apropulsive gait. The goal of the surgery was simply to give the patient a stable foot on which to ambulate. All the fusion sites and grafts were also augmented with Symphony platelet-rich plasma (Symphony platelet concentration system, Depuy Spine, Raynham, Massachusetts). Bone grafting with DBM is useful in reconstructive surgery to ultimately provide anatomic alignment, restore function, or augment/change the biomechanics of the foot and ankle. Because of the lack of structural integrity, DBM should be used in conjunction with a transplant or implant displaying mechanical strength. DBM can augment cortical grafts used for bridging gaps or defects and lengthening procedures by increasing the connectivity of the structural graft with the host bone. Another useful application is the biologic boost that some of the graft’s properties can provide to a patient who has less-than-ideal physiology. Because DBM has higher concentrations of available BMP, it can aid in the incorporation of other grafts. Grafts should be considered when reconstructing traumatic defects such as a comminuted calcaneal fracture and when the host bone can be buttressed and the void filled with DBM. Studies have shown that cancellous DBM solidifies the void faster and is replaced with structural host bone at a quicker rate than structural/cortical graft. Synthetic cements could be used but lack the inductive properties and matrix proteins of DBM. Other uses include but are not limited to delayed unions, nonunions, packing joints for arthrodesis, filling resected cysts, and filling gaps of debrided infected bone.
Listed demineralized bone matrix manufacturers Accell DBM100 (IsoTis OrthoBiologics, Irvine, California) Accell ConnexusTM (IsoTis OrthoBiologics) AlloCraftTM DBM (Stryker Howmedica Osteonics, Allendale, New Jersey) Allogro (AlloSource, Denver, Colorado) AlloMatrix Putty (Wright Medical Technology, Arlington, Tennessee) DBX Putty (Synthes, Paoli, Pennsylvania) DynaGraft (GenSci Regeneration Sciences and Innova Technologies Corp., Toronto, Ontario, Canada) Grafton (Osteotech) InterGroTM Putty (Interpore Cross International, Irvine, California) Opteform (Exactech) Optium (LifeNet, Virginia Beach, Virginia) Osteofil (Regeneration Technologies)
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References [1] Benzel E. Spine surgery: Techniques, complication avoidance, and management. 2nd edition. Volume 1. New York7 Elsevier, Churchill, Livingstone; 2005. [2] Van Meekeren J. Heel-en geneeskonstige aanmerkingen [Comments on healing a skull defect]. Amsterdam7 Comelijn; 1668. [3] Barth A. Histologische unterschungren uber Knochen-implantationer [Histologic understanding of improved bone implants]. Beitr Pathol Anat 1895;17:65 – 142. [4] Urist M, McLean F. Osteogenic potency and new-bone formation by induction in transplants to the anterior chamber of the eye. J Bone Joint Surg Am 1952;34:443 – 67. [5] Urist M. Bone formation by autoinduction. Science 1965;150:893 – 9. [6] Ragni P, Lindholm TS. Interaction of allogenic DBM and porous HA bioceramics in lumbar interbody fusion in rabbits. Clin Orthop 1991;272:292 – 9. [7] Han B, Tang B, Nimni ME. Quantitative and sensitive in vitro assay for osteoinductive activity of demineralized bone matrix. J Ortho Res 2003;4:648 – 54. [8] Atti E, Abjornson C, Diegmann M, et al. High resolution X-ray computed tomography as a technique to study osteoinductivity of demineralized bone matrix. Proceedings of the NASS 18th annual meeting. Spine J 2003;3:120S. [9] Yoo J, Birkedal J, Chang S, et al. Spinal arthrodesis using a demineralized bone/hyaluronan matrix and bone marrow. Proceedings of the NASS 18th annual meeting. Spine 2003;3:143S. [10] Edwards JL. In vivo osteoinductivity of two human DBM materials: comparative histopathologic evaluation at 14 and 28 days. London: IsoTis OrthoBiologics; 2003 [Publication #AW-0166.0]. [11] Edwin L, Blitch IV, Nicholas M, et al. McGlamry’s comprehensive textbook of foot and ankle surgery, vol. 2. 3rd edition. Philidelphia: Lippincott Williams & Wilkins; 2001. p. 1466–88. [12] Aro H, Eerola E, Aho AJ, et al. Tissue oxygen tension in externally stabilized tibial fractures in rabbits during normal healing and infection. J Surg Res 1984;37(3):202 – 7. [13] Kado K, Gambetta L, Perlman M. Uses of Grafton for reconstructive foot and ankle surgery. J Foot Ankle Surg 1996;35(1):59 – 66. [14] Martin Jr GJ, Boden SD, Titus L, et al. New formulations of demineralized bone matrix as a more effective graft alternative in experimental posterolateral lumbar spine arthrodesis. Spine 1999; 24:637 – 45.
Demineralized Bone Matrix: Basic Science and Clinical Applications Douglas J. Pacaccio, DPM, Stephen F. Stern, DPMT Podiatric Surgery Section, Department of Orthopaedics, Inova Fairfax Hospital, 3300 Gallows Road, Falls Church, VA 22042, USA
Demineralized bone matrix (DBM) is one of many modalities that can be useful in foot and ankle surgery when used appropriately. Any surgeon who performs osseous work, whether it is fracture fixation or major reconstructive surgery, always faces the ultimate task of achieving bony union. Obtaining successful fusion of bone requires several factors be present and working in concert with one another. The multiple variables include local environment of the fusion site, systemic factors, physiologic factors, mechanical factors, and at times, fusion enhancers.
Basic science Despite relatively good results across most patient populations, there are times when a surgeon needs every advantage afforded to achieve the end goal of bony healing. Some examples include large defects in the desired healing site and patients who have osteoporosis, osteopenia, Charcot’s neuroarthropathy, or other conditions in which bony resorption occurs at a higher rate than bony deposition. Another patient population shown to be at a disadvantage for acute bone healing is smokers. Systemic nicotine is well known to retard osteogenesis and inhibit vascular ingrowth. Smoke extracts also cause calcitonin resistance, increase fracture end resorption, and decrease osteoblast function [1]. Other systemic factors include drugs like many chemotherapeutic agents and particularly nonsteroidal anti-inflammatory drugs. Several studies in the literature have shown that indo-
T Corresponding author. E-mail address: [email protected] (S.F. Stern). 0891-8422/05/$ – see front matter D 2005 Elsevier Inc. All rights reserved. doi:10.1016/j.cpm.2005.07.001 podiatric.theclinics.com
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methacin significantly inhibits spinal fusion compared with saline controls in rat models. The physiology of osteoporosis manifests changes in bone marrow quality and bone turnover. There are less osteogenic stem cells in the bone of elderly patients, which equals a decrease in the density of bone, further making the mechanical stability of fixation more difficult. Hormones can also play a role in adjusting the physiology of bone formation. Many growth hormones, by way of somatomedins, increase bone formation. Corticosteroids oppositely inhibit osteoblast differentiation and decrease matrix synthesis while weakening collagen bonds. Local factors include surface area of the fusion site, how well the ends are in apposition, and purposeful dissection preserving adequate blood supply. It is in these circumstances that various bone grafts and graft substitutes/enhancers are gaining favor in major reconstructive foot and ankle surgery along with standard procedures in higher risk patients. The first reported bone graft was canine skull xenograft used to repair a defect in a soldier’s skull. This graft was done in 1668 by Dutch surgeon Job Van Meekeren. On attempted removal of the graft, it was noted that the graft had completely healed the defect [2]. Grafts are often used to enhance either some or all of the physiologic and structural factors previously mentioned. Ideally, a graft has the same properties of healthy host bone, including the biology of bone repair coupled with mechanical stability. Depending on the type of graft, it can be considered an implant or simply a host-tissue transplant. Biodegradable grafts are preferred because they incorporate into the host bone and allow for remodeling as necessary for maximum functional outcomes. These grafts also eliminate stress risers produced by modulus gradients between host bone and permanent materials. There are three basic types of biodegradable bone grafts. Autograft is bone harvested from the recipient’s own body. Allograft is bone harvested from a donor of the same species. Xenograft is bone harvested from of donor of a different species, usually bovine or porcine. Xenograft is rarely, if ever, used today because of its antigenicity and the high rate of rejection seen in the past. A bone graft may have one, two, or all of the three basic properties of bone regeneration: osteogenicity, osteoinduction, and osteoconduction. Osteogenicity refers to the presence of viable osteoprogenitor cells within the graft. These cells aid in the early stages of bridging and uniting graft and host bone. Only autograft and bone marrow are osteogenic. Osteoinduction denotes the capability of stimulating pluripotential stem cells to differentiate into functioning osteogenic cells such as osteoblasts and osteoclasts. Allograft has osteoinductive and osteoconductive properties. Osteoconduction implies graft capacity to facilitate neovascularization and infiltration of the graft by osteogenic precursor cells by way of ‘‘creeping substitution.’’ Essentially, these types of grafts act only as scaffolding for new bone to bridge across. Examples of strictly osteoconductive grafts are calcium phosphate, ceramics, coral, and collagen. A graft’s net biologic contribution to any fusion site resides in it’s ability to display any or all of the three basic properties of bone regeneration, osteogenicity, osteoinduction, and osteoconduction. The ability to use inherent osteoblasts in
basic science and clinical applications of dbm
601
early phases plus the capability to activate host osteoprogenitor cells coupled with facilitation of new bony ingrowth affect any graft’s viability. As the surgeon progresses the span from autograft to synthetic structural grafts, these biologic factors are decreased. The closer the graft is to having all three potentials, the less the graft relies on the host environment for replacement of graft tissue with host tissue. DBM is allogenic bone graft that has been processed into a putty or gel form. Although allogenic bone graft has been used by surgeons for the better part of 100 years, Barth [3] first proposed the concept of osteoinduction in 1893. Urist and McLean [4,5] further described its modern osteoinductive properties with relation to bone morphogenic proteins (BMP) in 1952. DBM is thought to possess more inductive properties than regular allograft because growth factor availability is increased after the demineralization process. DBM is different from cortical graft because it is mainly cancellous bone tissue. Cortical graft is desirable for structural support but has decreased osteogenicity relative to cancellous bone or marrow bone. Cortical bone has less surface area per unit weight and, therefore, less connectivity. The term connectivity describes the ability of an osteoconductive graft material to be ‘‘connected’’ to host bone and relates to the surface area available in the local healing environment for incorporation into the fusion mass. Because of the increased density of cortical bone, the rate of vascular ingrowth is slower. Although this construct presents increased relative mechanical strength at the time of surgery, it lags in mechanical strength and stability near the incorporation/remodeling phase of healing. The surgeon must weigh the delicate balance between the need for immediate structural support and the higher rate of incorporation that increased connectivity provides. Most often, regarding reconstructive forefoot and rearfoot surgery, a combination of cortical graft and DBM is preferred to maximize both structural support and connectivity of the fusion site. There are relatively scarce studies in the literature comparing DBM with autograft, synthetic graft, and other forms of allograft in foot and ankle surgery. Most of the literature on DBM and BMP is related to spine surgery because this is probably where these products are most commonly used. It was generally thought that autograft incorporates into host site faster than allograft, but Ragni and Lindholm [6] showed that DBM with hydroxyapatite (HA) demonstrated the quickest fusion rate compared with autograft and DBM or HA alone. These researchers used a rabbit interbody spine fusion model and ultimately concluded that at 6 months, the autograft results were comparable to the composite in fusion and mechanical strength. These findings may be the result of increased availability of BMP-saturated DBM (secondary to the demineralization process) potentiated by the increased connectivity of the graft site. There are several studies showing that DBM has a slightly higher concentration of BMP density than autograft. A study designed by Han and colleagues [7] showed that at 28 days, postintramuscular implantation of varying concentrations of DBM in an athymic nude rat model showed a direct correlation between DBM and mineralization of tissue. The implants were analyzed radiographically for mineral density. They were also analyzed for
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alkaline phosphatase activity on pluripotent myocytes. In both cases, the 100% active DBM displayed the most osteoinductive response, with each decrease in active DBM proportionally decreasing inductivity. These investigators described their results as having a ‘‘proportional osteoinductivity’’ response. Atti and colleagues [8] performed a similar study and reported the same conclusion. Yoo and coworkers [9] also showed that concentration of DBM improved osteoinductivity. They used DBM and bone marrow aspirate (BMA) in groups of skeletally mature rabbits. Two groups were implanted with varying amounts of DBM/HA/BMA, one group was implanted with DBM/HA only, and the two control groups were autograft only and BMA only. Of all the groups, it was found that those with the highest concentration of DBM, regardless of the BMA, showed the highest bone volumes in the fusion site. These investigators concluded that the amount of bone deposition was directly dependent on the amount of DBM present in the fusion site. Edwards [10] compared the osteoinductivity between a human DBM product with 100% DBM and one with 17% DBM in an athymic mouse population. Intramuscularly implanted DBM yielded ectopic bone samples that were sectioned at 14 days and 28 days. Histologic analysis revealed that 100% DBM outperformed 17% DBM in bone formation at both intervals. Graft healing occurs in five stages relatively similarly to simple fracture healing [11]. The first and second stages are marked by massive hemorrhage and inflammation, which occurs at the site of surgery as a result of surgical trauma. At these phases, the graft is infiltrated by polymorphonuclear leukocytes. During this transition, osteocytes die off but surface osteoblasts produce bone. Electronegativity gradients develop and create a physiologic environment conducive to bone healing. The gradient reduces pH and decreases oxygen tension. Relatively high oxygen tension signals mesenchymal stem cells to differentiate into osteoblasts and osteoclasts. Relatively low oxygen tension produces fibroblasts and chondroblasts from pleuripotential stem cells. Both scenarios occur in an environment with oxygen tension that is decreased from baseline. Aro and colleagues [12] showed that normal bone healing occurs in a relatively hypoxic environment. As the influx of inflammatory cells arrives at the fracture/ osteotomy site, the increased acidity decreases oxygen tension and stimulates osteoprogenitor stem cells. In a simple fracture, these osteoprogenitor stem cells are found mainly in the endosteum and cambium layer of periosteum. Marrow and host connective tissue also contains mesenchymal stem cells. It is at this phase that BMP-carrying DBM may aid in bony fusion by signaling stem cells, mainly through the transforming growth factor b superfamily, to become osteoprogenitor cells and begin the process of creating new bone. The relative early impact of BMP on the natural healing process possibly explains why autograft osteogenicity may have only little advantage in early healing over allograft. The advantage of increased concentration of BMP present in DBM, especially discernable in the previously cited studies, results in DBM composites achieving faster fusion. The third phase involves neovascularization of the graft. Host vessels invade the graft and provide nutrients for the following phase. This
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phase of healing can occur as soon as 2 days in cancellous (inductive) graft materials and slower in cortical graft. At the fourth phase, creeping substitution occurs as osteoblasts line the graft trebeculae and lay down osteoid. Creeping substitution is the term coined to describe the osteoclast cutting cones infiltrating necrotic bone followed by osteoblasts. At the fifth phase, the graft becomes remodeled from woven bone to lamellar bone, which has lower density of osteocytes and more parallel/organized collagen orientation. In addition, the medullary canal is created within the graft. Ideally, the host bone eventually replaces the graft and any entrapped graft is resorbed. Allograft bone is prepared for use by freezing, freeze-drying, or decalcifying. Simple freezing of allograft bone is known to be more antigenic than freezedrying or decalcifying. All forms of preparation destroy the viability of living osteocytes within the graft, thus removing any osteogenic properties. DBM is decalcified and maintains its inductive and conductive properties by preserving growth factors, BMPs, collagen, and matrix proteoglycans. The specimen is harvested no greater than 24 hours post mortem. Harvesting is done after performing a detailed medical history of the donor and donor’s family. The donor’s blood is tested for hepatitis A antibody, hepatitis B surface antigen, hepatitis B core antibody, hepatitis C antibody, HIV-1 virus antibodies, HIV direct antigen, human T-cell lymphoma virus-1 antibody, syphilis, and aerobic/anaerobic cultures. The surrounding tissues are also tested for surface and medullary contaminants (aerobic and anaerobic). The sample is irradiated with high-dose gamma radiation, and packaged and tested for sterility. Following initial testing, the specimen is stripped of any soft tissue and the bone is transported in bacitracin and polymyxin B solution. It is important to know that trace amounts of these antimicrobials remain in the final product, and patient hypersensitivity should be considered an absolute contraindication. The bone is milled into gross particle size when blood, marrow, and lipids are removed by washing with distilled water. The specimen is placed in an ultrasound bath before being cleared of lipids with 70% ethanol. The particles are flushed again with water and milled again to their final size. When particles reach their final size, they are demineralized in 0.6 M hydrochloric acid for 3 hours. The sample loses apatite crystals but retains its matrix calcium. The product is rinsed again with water and buffered with 0.1 M phosphate solution to the final pH. The sample undergoes two separate rounds of lyophilization (freeze-drying) to enable storage at room temperature. The sample is usually placed in its final packaging between rounds. Testing of room sterility, moisture, and calcium content finalizes the process. This process complies with the United States Pharmacopeia standards and the American Association of Tissue Banks [1,13]. Depending on the manufacturer, DBM comes in several forms. Grafton (Osteotech, Eatontown, New Jersey) was originally produced in a glycerol gel carrier. It is now available in putty form or as flexible sheets, which have been shown to produce higher fusion rates [14]. Osteofil (Regeneration Technologies, Alachua, Florida) is set in a hydrogel matrix. Opteform (Exactech, Gainesville, Florida) comes in a formable thermoplastic inert biodegradable carrier.
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Clinical applications Kado and colleagues [13] described various uses of Grafton DBM in the foot and ankle in 1996. Among the suggested applications are arthrodesis of the following joints: pantalar, ankle, triple (talonavicular, subtalar, and calcaneocuboid), Lisfranc’s, naviculocuneiform/intercuneiform, subtalar, and metatarsalphalangeal. They also performed osteotomies of the calcaneus (Silver, Evans) and midfoot (Cole). Other applications included tibiofibular fracture, metatarsal gunshot wound, Jones fracture nonunion, replaced donor site of autograft from calcaneus, medullary canal defect, first metatarsal cyst, and naviculocuneiform nonunion. These investigators reported good results and ease of application with the gel form of Grafton to fill gaps in bony fusion sites. Fig. 1 shows postoperative radiographs of a 72-year-old woman who underwent a triple arthrodesis and was found to have a fairly atrophic and soft navicular. She was noted to be osteopenic preoperatively, and several fixation modalities were requested to be present in the operating room as backup, including external fixation rails. Note that the first naviculocuneiform joint was also fused, which served to increase compression across the talonavicular joint and to plantarflex the medial column from elevatus to plantar grade. The doctor in residence, Dr. D. Pacaccio, and the attending physician assessed the medial column intraoperatively and agreed on the hypermobility present. It was believed that this fusion achieved adequate stability without locking up all the motion in the medial column by leaving the first metatarsal cuneiform joint intact. Washers
Fig. 1. (A,B) Triple arthrodesis with medial column stabilization in grossly unstable flat-foot deformity. Tricortical subtalar joint allograft with minimal gapping secondary to DBM implantation.
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were used with several of the screws because the bone was relatively soft. Femoral neck allograft was placed in the subtalar joint fusion site and the calcaneocuboid joint. It was decided intraoperatively to use Grafton DBM in the subtalar, calcaneocuboid, and talonavicular fusion sites and in the sinus tarsi. This application of DBM is a good one, in that it should help to create a bony bridge, adding to the stability of the fusion. The patient had an extremely planovalgus foot preoperatively and has a fairly apropulsive gait. The goal of the surgery was simply to give the patient a stable foot on which to ambulate. All the fusion sites and grafts were also augmented with Symphony platelet-rich plasma (Symphony platelet concentration system, Depuy Spine, Raynham, Massachusetts). Bone grafting with DBM is useful in reconstructive surgery to ultimately provide anatomic alignment, restore function, or augment/change the biomechanics of the foot and ankle. Because of the lack of structural integrity, DBM should be used in conjunction with a transplant or implant displaying mechanical strength. DBM can augment cortical grafts used for bridging gaps or defects and lengthening procedures by increasing the connectivity of the structural graft with the host bone. Another useful application is the biologic boost that some of the graft’s properties can provide to a patient who has less-than-ideal physiology. Because DBM has higher concentrations of available BMP, it can aid in the incorporation of other grafts. Grafts should be considered when reconstructing traumatic defects such as a comminuted calcaneal fracture and when the host bone can be buttressed and the void filled with DBM. Studies have shown that cancellous DBM solidifies the void faster and is replaced with structural host bone at a quicker rate than structural/cortical graft. Synthetic cements could be used but lack the inductive properties and matrix proteins of DBM. Other uses include but are not limited to delayed unions, nonunions, packing joints for arthrodesis, filling resected cysts, and filling gaps of debrided infected bone.
Listed demineralized bone matrix manufacturers Accell DBM100 (IsoTis OrthoBiologics, Irvine, California) Accell ConnexusTM (IsoTis OrthoBiologics) AlloCraftTM DBM (Stryker Howmedica Osteonics, Allendale, New Jersey) Allogro (AlloSource, Denver, Colorado) AlloMatrix Putty (Wright Medical Technology, Arlington, Tennessee) DBX Putty (Synthes, Paoli, Pennsylvania) DynaGraft (GenSci Regeneration Sciences and Innova Technologies Corp., Toronto, Ontario, Canada) Grafton (Osteotech) InterGroTM Putty (Interpore Cross International, Irvine, California) Opteform (Exactech) Optium (LifeNet, Virginia Beach, Virginia) Osteofil (Regeneration Technologies)
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References [1] Benzel E. Spine surgery: Techniques, complication avoidance, and management. 2nd edition. Volume 1. New York7 Elsevier, Churchill, Livingstone; 2005. [2] Van Meekeren J. Heel-en geneeskonstige aanmerkingen [Comments on healing a skull defect]. Amsterdam7 Comelijn; 1668. [3] Barth A. Histologische unterschungren uber Knochen-implantationer [Histologic understanding of improved bone implants]. Beitr Pathol Anat 1895;17:65 – 142. [4] Urist M, McLean F. Osteogenic potency and new-bone formation by induction in transplants to the anterior chamber of the eye. J Bone Joint Surg Am 1952;34:443 – 67. [5] Urist M. Bone formation by autoinduction. Science 1965;150:893 – 9. [6] Ragni P, Lindholm TS. Interaction of allogenic DBM and porous HA bioceramics in lumbar interbody fusion in rabbits. Clin Orthop 1991;272:292 – 9. [7] Han B, Tang B, Nimni ME. Quantitative and sensitive in vitro assay for osteoinductive activity of demineralized bone matrix. J Ortho Res 2003;4:648 – 54. [8] Atti E, Abjornson C, Diegmann M, et al. High resolution X-ray computed tomography as a technique to study osteoinductivity of demineralized bone matrix. Proceedings of the NASS 18th annual meeting. Spine J 2003;3:120S. [9] Yoo J, Birkedal J, Chang S, et al. Spinal arthrodesis using a demineralized bone/hyaluronan matrix and bone marrow. Proceedings of the NASS 18th annual meeting. Spine 2003;3:143S. [10] Edwards JL. In vivo osteoinductivity of two human DBM materials: comparative histopathologic evaluation at 14 and 28 days. London: IsoTis OrthoBiologics; 2003 [Publication #AW-0166.0]. [11] Edwin L, Blitch IV, Nicholas M, et al. McGlamry’s comprehensive textbook of foot and ankle surgery, vol. 2. 3rd edition. Philidelphia: Lippincott Williams & Wilkins; 2001. p. 1466–88. [12] Aro H, Eerola E, Aho AJ, et al. Tissue oxygen tension in externally stabilized tibial fractures in rabbits during normal healing and infection. J Surg Res 1984;37(3):202 – 7. [13] Kado K, Gambetta L, Perlman M. Uses of Grafton for reconstructive foot and ankle surgery. J Foot Ankle Surg 1996;35(1):59 – 66. [14] Martin Jr GJ, Boden SD, Titus L, et al. New formulations of demineralized bone matrix as a more effective graft alternative in experimental posterolateral lumbar spine arthrodesis. Spine 1999; 24:637 – 45.