Keys To Optimal Selection Of Orthobiologics
Given the adjunctive potential of orthobiologics in facilitating bone healing, these authors offer a closer look at osteoconductive agents such as calcium sulfate and osteoinductive agents such as PRP and demineralized bone matrix, and the emerging literature on these modalities.
The musculoskeletal system has the extraordinary ability to regenerate itself. This property is shared with only a couple of systems (the liver and the hematopoietic system) in the human body. Bones are able to reconstitute into the same shape and size, and are able to withstand the same load as pre-injured bone. The ability to harness this mechanism has implications for reconstructive foot and ankle surgery.
The reality is that bone healing following elective surgery or trauma does not always go as expected. Complications such as delayed union, malunion and nonunion can occur. There are many factors that can contribute to a nonunion including: the presence of systemic diseases such as diabetes mellitus; pharmaceuticals that can adversely affect bone healing; infection; inadequate fixation; poor vascularity; bone and soft tissue defects; smoking; and nutritional deficiencies.
There has been an explosion in new technologies available to enhance bone healing in foot and ankle surgery. Not only have superior fixation constructs been engineered but there has also been an increase in the number and types of orthobiologics in the marketplace. Today, a myriad of commercially available bone grafts, bone graft extenders and osteobiologics have been developed for day-to-day use. Accordingly, let us review the basic science and take a closer look at the clinical application of orthobiologics as they apply to foot and ankle surgery.
An Overview Of The Mechanisms Of Orthobiologic Substrates
Osteoconduction is the process by which a biologic scaffold provides the framework for passive ingrowth of mesenchymal stem cells (MSCs), angiogenesis and the migration of bioactive signaling molecules.
Osteoinduction is the mechanism by which mesenchymal stem cells are recruited to the surgical site and activated to differentiate into chondroblasts and osteoblasts. This pathway leads to new bone formation through endosteal ossification. This complex pathway is mediated by soluble growth factors including bone morphogenetic proteins (BMPs), platelet-derived growth factor (PDGF), fibroblast growth factor (FGF) and vascular endothelial growth factor (VEGF).
Osteogenesis comprises the pathway in which mesenchymal stem cells, osteoblasts and osteocytes are transplanted locally or from another site for the purpose of osteosynthesis. This is achieved by utilizing autogenous graft from the host or through a bone marrow transplant.
Osteopromotion involves the utilization of agents to enhance bone healing that is already taking place.
Pertinent Insights On Osteoconductive Agents
Osteoconductive orthobiologics are scaffolds that have similar structural integrity of cancellous bone but lack the combination of osteoinduction and osteogeneic properties of true cancellous autograft. Osteoconductive agents serve as scaffolds on which osteoblasts can lay bone or as a mineral repository for cartilaginous calcification following their own resorption. These scaffolds passively allow ingrowth of host capillaries, perivascular tissue and mesenchymal stem cells, thereby fulfilling a permissive role in allowing new bone growth.1 These bone substitutes are available in powders, putty, pellets and as implant coatings for joint prostheses.
Advantages of osteoconductive substitutes are the fact that they are readily available, relatively inexpensive and serve a role in void filling. There is also an absence of donor site morbidity and viral transmission. The main disadvantage of these calcium compounds is the limited or lack of biological healing potential.2 Examples of osteoconductive orthobiologics include demineralized bone matrix (DBM), which is both osteoconductive and osteoinductive, calcium phosphate, and calcium sulfate.
Calcium sulfate is available in many forms. These sulfates are available as hard pellets and injectable fluids that harden in vivo. One can load calcium sulfate with antibiotic to help treat osteomyelitis. In a prospective study of 25 patients requiring debridement of long bone infection, McKee and colleagues found that infection was eradicated in 23 patients.3 Moreover, study authors noted unions in 14 out of 16 previous non-union patients with the use of tobramycin-impregnated calcium sulfate. Substantiating the claim that sulfate compounds are highly effective bone defect fillers, Gitelis and coworkers were able to attain healing in 21 out of 23 patients with bone defects, using calcium sulfate either in combination with demineralized bone matrix or in isolation.4 Demineralized bone matrix and calcium sulfate also remain the most rapidly absorbed bone substitutes.
Calcium phosphate substitutes are calcium salt compounds made up of calcium ions and organophosphates. Most of these compounds are available as a mixture of calcium salts and few are available as pure formulations.2 Compounds used in these bone grafts consist of mono-, di-, tri- and tetracalcium phosphates. Surgeons frequently use hydroxyapatite in its alpha and beta forms. Tricalcium phosphate remains one of the most commonly used osteoconductive substrates.5 Cements are popular because they are effective in filling bone defects and provide structural resistance to compression. However, the risks of site migration and peripheral tissue damage remain concerns.5
In general, unique advantages of calcium phosphates include osteointegration, slow biodegradation and compressive strength. In cases in which osteoinduction is also desired, surgeons have the option of using composite grafts that combine osteoinductive factors with a calcium phosphate matrix. These compounds provide mechanical support in acute settings but they lack the biological ability to facilitate healing in chronic defects.5 Some of these grafts reportedly have up to ten times the compressive strength of cancellous bone. However, Roberts and Rosenbaum point out that these assertions do not take into account tensile and shear forces, which, along with compression, occur in vivo.2
There is no hard evidence that suggests osteoconductive orthobiologics reliably and reproducibly facilitate bone defect healing in humans.5 However, a number of researchers have reported clinical success. In a prospective cohort study of 32 patients with 36 joint depression calcaneal fractures, Schildhauer and colleagues found that calcium phosphate cement in conjunction with standard open reduction internal fixation allowed patients to bear weight as early as three weeks.6
Surgeons can use osteoconductive orthobiologics as void fillers, infection eradicators (in combination with antibiotics) and graft expanders when they combine them with autogenous grafts or bone morphogenetic proteins. Researchers are currently looking at the efficacy of adding collagen, growth factors and mesenchymal stem cells to augment the biological activity of these products, and there is a growing need for further studies of these products in the lower extremity.
A Primer On Platelet-Rich Plasma
Platelet-rich plasma (PRP) is a volume of fractionated plasma of the patient’s blood that has platelet concentrations above normal levels in the body (150,000/μL-350,000/μL).7,8 In the last decade, platelets have gained intense popularity as an adjunctive treatment for musculoskeletal injuries.9
Specifically, platelets originate within the bone marrow from megakaryocytes. Circulating in the blood, they are non-nucleated, colorless, discoid-shaped cells that carry granules (α, δ, λ).10,11 Platelets are activated and aggregate together shortly after tissue damage with the alpha (α) granules releasing many proteins. Within 10 minutes of aggregation, platelets begin secreting these proteins and more than 95 percent are released within one hour.7,11 The most relevant ones secreted for healing are insulin-like growth factor (ILGF), epidermal growth factor (EGF), transforming growth factor (TGF-β1), PDGF, FGF and VEGF among many others.10,12-14
This complex signaling cascade ultimately leads to recruitment of mesenchymal stem cells and fibroblasts, collagen formation, angiogenesis and smooth muscle formation.14-16 The ideal amount of PRP is unknown but a minimum of 1,000,000/μL is required for tissue healing with no benefit observed over approximately five times normal levels.7,8
Clinicians obtain PRP by collecting the patient’s blood from a vein and mixing it with an anticoagulant. One then puts the blood into a centrifuge, where it is separated into a top acellular plasma layer (platelet-poor plasma), a middle buffy coat layer (PRP), and a bottom erythrocyte layer (red blood cells). Then the clinician collects the PRP with a syringe or by an automated system. Finally, PRP activates via the addition of calcium chloride or thrombin among other factors.17
Dohan and coworkers categorized platelet concentrates into four types, relying on fibrin and leukocyte content.17 The first type is pure platelet rich plasma, which is poor in leukocytes. The second is leukocyte rich PRP. The third type is pure platelet rich fibrin, which only encompasses Fibrinet (Vertical Spine).
The application of PRP for bone healing in the foot and ankle has had some promising results. Two randomized controlled trials (RCTs) found the combination of bone chips with platelet gel and/or bone marrow stromal cells increases the ostegenetic potential of the bone chips, and may be valuable in treating patients with difficulties in bone healing and patients with large bone deficits.18,19 Another RCT looking at the treatment of displaced intra-articular calacneal fractures compared three treatment groups.20 The first group received allograft alone. The second group received allograft and PRP. The third group received autograft plus PRP. The follow-up ranged from 24 to 72 months postoperatively. The researchers went on to find that the groups utilizing PRP had significantly superior outcomes in comparison to the group that received an allograft alone.20
What The Literature Reveals About Demineralized Bone Matrix
Demineralized bone matrix is a family of commercially available products produced from morselized corticocancellous bone, which is extracted from human cadavers.21 This human allograft is washed, deminerialized with organic solvents or acids, dried, prepared and sterilized.5 It is intrinsically osteoinductive and, to a lesser extent, osteoconductive.22 Researchers have shown that the osteoinductivity of demineralized bone matrix is dependent upon the demineralization process and those products washed with hydrochloride (HCl) are superior to those washed with other acids.5,22,23
At its core, demineralized bone matrix is comprised chiefly of collagen (93 percent), which provides its osteoconductive surface. Soluble proteins such as bone morphogenetic proteins and other synergistic proteins (PDGF, IGF, TGF and FGF) give demineralized bone matrix its inherent osteoinductivity and comprise approximately 5 percent of its makeup. The remaining 2 percent is made of residual mineralized matrix.24,25
It is important to emphasize that demineralized bone matrix does not directly induce the formation of bone in subcutaneous or submuscular tissues.24 Demineralized bone matrix induces mesenchymal stem cells to differentiate into chondroblasts, not osteoblasts. This chondrogenesis process differs from that of classical endochondral bone formation. Although cartilage forms, resorbs and is eventually replaced by bone, the bone formation facilitated by demineralized bone matrix occurs only after the cartilage has been resorbed and not at the same time as resorption as is the case in endochondral bone formation.24-27
All demineralized bone matrix is not created equally.28 Donor tissue is not pooled in the United States and each lot or batch of demineralized bone matrix derives from a single donor. Along with donor-to-donor variation is the proprietary nature of each demineralization process, which is neither widely published nor regulated.29 Once demineralized bone is extracted from donor bone, it is in a particulate powder form, which is easily subject to static charge which can make containment and delivery challenging.
Accordingly, demineralized bone matrix is combined with other carriers (e.g. glycerol, gelatin, calcium sulfate, hyaluronic acid, lecithin or poloxamer) to make handling and delivery easier. In such mixtures, a significant portion of the compound is comprised of carrier material (~85 percent carrier, ~15 percent demineralized bone matrix).24 Many different forms (e.g. gel, putty, chips) are commercially produced. Accordingly, there are significant variations in concentrations of bone morphogenetic proteins when comparing preparations, even between lot numbers of the same brand.21,28
What You Should Know About Bone Morphogenetic Proteins
Bone morphogenetic proteins are a group of bioactive molecules that have been the subject of intense research over the last 50 years. Since their discovery, research has revealed increasing indications in the clinical setting. Bone morphogenetic proteins can be produced in large quantities with recombinant DNA technology and cloning techniques.30 The ability to produce bone morphogenetic proteins in the lab has been instrumental in making them commercially available for patient care.30-32
Bone morphogenetic proteins are members of the tumor growth factor beta superfamily of molecules. These molecules function within complicated signaling pathways that modulate differentiation of mesenchymal stem cells and osteogenesis.33 BMP 2, 4, 7, 9 and 14 have shown the most promise in promoting bone healing. These complex molecules are known to incite the production of cartilage, new bone formation and angiogenesis.34,35
Furthermore, BMP-2 and BMP-7 have shown the most promise to foot and ankle surgeons due to their direct influence on angiogenesis.33 BMP-2 is of particular interest due to its abilities to promote neovascularization and induce the differentiation of mesenchymal stem cells into osteoblasts.33 This powerful molecule has been well studied in the orthopedic literature. Large multicenter randomized trials have led to FDA approval for the use of BMP-2 in acute open tibial fractures.33
BMP-7 has the ability to promote new blood vessel formation and has been shown to have strong osteoinductive properties. Recent level 1 studies have demonstrated promising results in the orthopedic literature. Friedlander and colleagues demonstrate that BMP-7, when mixed with a collagen carrier, was as good as autogenous graft in the treatment of tibial non-unions.36 Furthermore, BMP-7 has been FDA approved for revision arthrodesis of the lumbar spine.33
The evidence on BMP is not all positive. Bone morphogenetic protein is a soluble substance that can diffuse away from its intended site of action and thus needs a carrier to be effective. Furthermore, BMP can cause the formation of ectopic bone, leading to painful soft tissue entrapments, and incite strong inflammatory reactions.37
While the current evidence is good, further studies are needed to assess the morbidities associated with the recombinant bone morphogenic proteins in foot and ankle surgery.
The skeletal system’s ability to heal itself is truly remarkable. Bone healing relies upon a complex interplay of intrinsic and extrinsic factors. These factors include a stable fixation construct, nutritional status and the presence of native growth factors and osteogenic cells. When this microenvironment is out of balance, non-union may result. Understanding the mechanisms of bone healing and the attributes of available orthobiologic substrates will give the foot and ankle surgeon the tools to enhance the musculoskeletal system’s innate ability to regenerate itself.
Drs. Richardson, Dix, Adeleke and Baca are residents within the Division of Foot and Ankle Surgery at the Western Pennsylvania Hospital in Pittsburgh.
Dr. Mendicino is in private practice with OhioHealth Orthopedic Surgeons in Hilliard, Ohio. He is a Fellow and Past President of the American College of Foot and Ankle Surgeons. Dr. Mendicino is board-certified in foot surgery and rearfoot and ankle surgery by the American Board of Podiatric Surgery.
Dr. Catanzariti is the Director of the Residency Training Program within the Division of Foot and Ankle Surgery at the Western Pennsylvania Hospital in Pittsburgh. He is a Fellow of the American College of Foot and Ankle Surgeons.
1. Southerland J, Alder D, Boberg J, Downey M, Nakra A, Rabjohn L, et al (eds): McGlamry’s Comprehensive Textbook of Foot and Ankle Surgery. 4th edition, Elsevier, Philadelphia, 2013.
2. Roberts TT, Rosenbaum AJ. Bone grafts, bone substitutes and orthobiologics: The bridge between basic science and clinical advancements in fracture healing. Organogenesis. 2012;8(4). (Epub ahead of print)
3. McKee MD, Wild LM, Schemitsch EH, Waddell JP. The use of an antibiotic- impregnated, osteoconductive, bioabsorbable bone substitute in the treatment of infected long bone defects: early results of a prospective trial. J Orthop Trauma. 2002;16(9):622-627.
4. Gitelis S, Piasecki P, Turner T, Haggard W, Charters J, Urban R. Use of a calcium sulfate-based bone graft substitute for benign bone lesions. Orthopedics. 2001;24(2):162-166.
5. Mckee MD. Management of segmental bony defects: the role of osteoinductive orthobiologics. J Am Acad Orthop Surg. 2006;14(10 Spec No.):S163-S167.
6. Schildhauer TA, Bauer TW, Josten C, Muhr G. Open reduction and augmentation of internal fixation with an injectable skeletal cement for the treatment of complex calcaneal fractures. J Orthop Trauma. 2000;14(5):309-17.
7. Marx RE. Platelet-rich plasma (PRP): what is PRP and what is not PRP? Implant Dent. 2001;10(4):225-8.
8. Marx RE. Platelet-rich plasma: evidence to support its use. J Oral Maxillofac Surg. 2004;62(4):498-96.
9. Mehta S, Watson JT. Platelet rich concentrate: basic science and current clinical applications. J Orthop Trauma. 2008;22(6):432–8.
10. Sampson S, Gerhardt M, Mandelbaum B: Platelet rich plasma injection grafts for musculoskeletal injuries: a review. Curr Rev Musculoskelet Med. 2008;1(3-4):165-174.
11. Harrison P, Cramer EM. Platelet alpha-granules. Blood Rev. 1993;7(1):52–62.
12. Broughton G 2nd, Janis JE, Attinger CE. Wound healing: an overview. Plast Reconstr Surg. 2006;117(Suppl 7):1e-S–32e-S.
13. Smith SE, Roukis TS. Bone and wound healing augmentation with platelet-rich plasma. Clin Podiatr Med Surg. 2009;26(4):559–88.
14. Eppley BL, Woodell JE, Higgins J. Platelet quantification and growth factor analysis from platelet-rich plasma: implications for wound healing. Plastic Reconstruct Surg. 2004;114(6):1502-1508.
15. Everts PA, Knape JT, Weibrich G, Schonberger JP, Hoffmann J, Overdevest EP, Box HA, van Zundert A: Platelet-rich plasma and platelet gel: a review. J Extra Corpor Technol. 2006;38(2):174-187.
16. Engebretsen L, Steff en K, Alsousou J, et al. IOC consensus paper on the use of platelet-rich plasma in sports medicine. Br J Sports Med. 2010; 44(15):1072-1081.
17. Dohan Ehrenfest DM, Rasmusson L, Albrektsson T: Classification of platelet concentrates: from pure platelet-rich plasma (P-PRP) to leucocyte- and platelet-rich fibrin (L-PRF). Trends Biotechnol. 2009; 27(3):158-167.
18. Savarino L, Cenni E, Tarabusi C, Dallari D, Stagni C, Cenacchi A, et al. Evaluation of bone healing enhancement by lyophilized bone grafts supplemented with platelet gel: a standardized methodology in patients with tibial osteotomy for genu varus. J Biomed Mater Res B Appl Biomater. 2006;76(2):364-72.
19. Dallari D, Savarino L, Stagni C, Cenni E, Cenacchi A, Fornasari PM, et al. Enhanced tibial osteotomy healing with use of bone grafts supplemented with platelet gel or platelet gel and bone marrow stromal cells. J Bone Joint Surg Am. 2007;89(11):2413-20.
20. Wei LC, Lei GH, Gao SG, Xu M, Jiang W, Song Y, et al. Efficacy of platelet-rich plasma combined with allograft bone in the management of displaced intra-articular calcaneal fractures: a prospective cohort study. J Orthop Res. 2012;30(10):1570-6.
21. Grabowski G, Cornett CA. Bone graft and bone graft substitutes in spine surgery: current concepts and controversies. J Am Acad Orthop Surg. 2013;21(1):51-60.
22. Johnson EE, Urist MR, Finerman GA. Bone morphogenetic protein augmentation grafting of resistant femoral nonunions. A preliminary report. Clin Orthop Relat Res. 1988;230:257-265.
23. Peterson B, Whang PG, Iglesias R, Wang JC, Leiberman JR. Osteoinductivity of commercially available demineralized bone matrix. J Bone Joint Surg Am. 2004;86-A(10):2243-2250.
24. Lee KJH, Roper JG, Wang JC. Demineralized bone matrix and spinal arthrodesis. Spine J. 2005;5(6 Suppl):217s-223s.
25. Martin GJ, Boden SD, Titus L, Scarborough NL. New formulations of demineralized bone matrix as a more effective graft alternative in experimental posterolateral lumbar spine arthrodesis. Spine. 1999;24(7):637–45.
26. Wang J, Glimcher M. Characterization of matrix-induced osteogenesis in rat calvarial bone defects: I. Differences in the cellular response to demineralized bone matrix implanted in calvarial defects and in subcutaneous sites. Calcif Tissue Int. 1999;65(2):156–65.
27. Wang J, Yang R, Gerstenfeld LC, Glimcher MJ. Characterization of demineralized bone matrix-induced osteogenesis in rat calvarial bone defects: III. Gene and protein expression. Calcif Tissue Int. 2000;67(4):314–20.
28. Watson JT. Overview of biologics. J Orthop Trauma. 2005;19(10 Suppl):S14-S16.
29. Pietrzak WS, Perns SV, Keys J, Woodell-May J, McDonald NM. Demineralized bone matrix graft: a scientific and clinical case study assessment. J Foot Ankle Surg. 2005;44(5):345-353.
30. Rihn JA, Gates C, Glassman SD, Phillips FM, Schwender JD, Albert TJ. The use of bone morphogenetic protein in lumbar spine surgery. J Bone Joint Surg Am. 2008; 90(9):2014-25.
31. Mendenhall S. Bone graft and bone substitutes. Orthopedic Network News. 2008:19(4):18-21. http://spine.orthopedicnetworknews.com/archives/onn194s6.pdf . Published November 10, 2008. Accessed August 5, 2013.
32. Obremskey WT, Marotta JS, Yaszemski MJ, Churchill LR, Boden SD, Dirschl DR. Symposium. The introduction of biologics in orthopaedics: issues of cost, commercialism, and ethics. J Bone Joint Surg Am. 2007; 89(7):1641-9.
33. Flynn JM. Fracture repair and bone grafting. In: Flynn JM (ed): OKU 10: Orthopaedic Knowledge Update. American Academy of Orthopaedic Surgeons, Rosemont, IL, 2011, pp. 11-21.
34. Milano F, van Baal JW, Buttar NS, Rygiel AM, de Kort F, DeMars CJ, et al. Bone morphogenetic protein 4 expressed in esophagitis induces a columnar phenotype in esophageal squamous cells. Gastroenterology. 2007;132(7):2412-21.
35. Kodach LL, Wiercinska E, de Miranda NF, Bleuming SA, Musler AR, Peppelenbosch MP, et al. The bone morphogenetic protein pathway is inactivated in the majority of sporadic colorectal cancers. Gastroenterology. 2008;134(5):1332-41.
36. Friedlaeder GE, Perry CR, Cole JD, Cook SD, Cierny G, Muschler GF, et al. Osteogenic protein-1 (bone morphogenetic protein-7) in the treatment of tibial nonunions. J Bone Joint Surg Am. 2001;83-A Suppl 1(Pt 2):S151-158.
37. Cahill KS, Chi JH, Day A, Claus EB. Prevalence, complications, and hospital charges associated with use of bone-morphogenetic proteins in spinal fusion procedures. JAMA. 2009;302(1):58-66.
38. Foster TE, Puskas BL, Mandelbaum BR, et al. Platelet-rich plasma: from basic science to clinical applications. Am J Sports Med. 2009;37(11):2259–72.
39. Bhanot S, Alex JC. Current applications of platelet gels in facial plastic surgery. Facial Plast Surg. 2002;18(1):27–33.
40. Aksahin E, Doğruyol D, Yüksel HY, Hapa O, Doğan O, Celebi L, Biçimoğlu A. The comparison of the effect of corticosteroids and platelet-rich plasma (PRP) for the treatment of plantar fasciitis. Arch Orthop Trauma Surg. 2012;132(6):781-5.
41. Martinelli N, Marinozzi A, Carnì S, Trovato U, Bianchi A, Denaro V. Platelet-rich plasma injections for chronic plantar fasciitis. Int Orthop. 2013;37(5):839-42.
42. Ragab EM, Othman AM. Platelets rich plasma for treatment of chronic plantar fasciitis. Arch Orthop Trauma Surg. 2012;132(8):1065-70.
43. Schepull T, Kvist J, Norrman H, Trinks M, Berlin G, Aspenberg P. Autologous platelets have no effect on the healing of human Achilles tendon ruptures: a randomized single-blind study. Am J Sports Med. 2011;39(1):38-47.
44. De Vos RJ, Weir A, Tol JL, Verhaar JA, Weinans H, van Schie HT. No effects of PRP on ultrasonographic tendon structure and neovascularisation in chronic midportion Achilles tendinopathy. Br J Sports Med. 2011;45(5):387-392.
45. De Jonge S, de Vos RJ, Weir A, van Schie HT, Bierma-Zeinstra SM, Verhaar JA, Weinans H, Tol JL. One-year follow-up of platelet-rich plasma treatment in chronic Achilles tendinopathy: a double-blind randomized placebo-controlled trial. Am J Sports Med. 2011 Aug;39(8):1623-9.
46. Martinez-Zapata MJ, Marti-Carvajal AJ, Sola I, Exposito JA, Bolibar I, Rodriguez L, et al. Autologous platelet-rich plasma for treating chronic wounds. Cochrane Database Syst Rev. 2012 Oct 17;10:CD006899. doi: 10.1002/14651858.CD006899.pub2.
47. Planinsek Rucigaj T, Lunder T. Stimulation of venous leg ulcers with thrombocytic growth factors: A randomized study. 17th Conference of the European Wound Management Association; Glasgow, Scotland; May 2-4, 2007. 2007:62 Abstract No. 93. Available at: http://ewma.org/fileadmin/user_upload/EWMA/pdf/conference_abstracts/2007...
48. Senet P, Bon FX, Benbunan M, Bussel A, Traineau R, Calvo F. Randomized trial and local biological effect of autologous platelets used as adjuvant therapy for chronic venous leg ulcers. J Vasc Surg. 2003;38(6):1342–8.
49. Stacey MC, Mata SD, Trengove NJ, Mather CA. Randomised double-blind placebo controlled trial of topical autologous platelet lysate in venous ulcer healing. Eur J Vasc Endovasc Surg. 2000;20(3):296–301.
50. Driver VR, Hanft J, Fylling CP, Beriou JM, Autologel Diabetic Foot Ulcer Study Group. A prospective, randomized, controlled trial of autologous platelet-rich plasma gel for the treatment of diabetic foot ulcers. Ostomy Wound Manage. 2006;52(6):68–70,72,74.
51. Kakagia D, Kazakos K, Xarchas K, Karanikas M, Georgiadis G, Tripsiannis G, et al. Synergistic action of protease-modulating matrix and autologous growth factors in healing diabetic foot ulcers. A prospective randomized trial. J Diabetes Complications. 2007;21(6):387–91.
52. Knighton DR, Ciresi K, Fiegel VD, Schumerth S, Butler E, Cerra F. Stimulation of repair in chronic, nonhealing, cutaneous ulcers using platelet-derived wound healing formula. Surg Gynecol Obstet. 1990;170(1):56–60.
53. Krupski WC, Reilly LM, Perez S, Moss KM, Crombleholme PA, Rapp JH. A prospective randomized trial of autologous platelet-derived wound healing factors for treatment of chronic nonhealing wounds: a preliminary report. J Vasc Surg. 1991;14(4):526–32.
54. Weed B, Davis MDP, Felty CL, Liedl DA, Pineda AA, Moore SB, Rooke TW. Autologous platelet lysate product versus placebo in patients with chronic leg ulcerations: a pilot study using a randomized, double-blind, placebo controlled trial. Wounds. 2004;16(9):273–82.
55. Anitua E, Aguirre JJ, Algorta J, Ayerdi E, Cabezas AI, Orive G, et al.Effectiveness of autologous preparation rich in growth factors for the treatment of chronic cutaneous ulcers. J Biomed Mater Res B Appl Biomater. 2008;84(2):415–21.
56. Bae HW, Zhao L, Kanim LE, Wong P, Delamarter RB, Dawson EG: Intervariability and intravariability of bone morphogenic proteins in commercially available bone matrix products. Spine. 2006;31(12):1299-1306.
57. Peterson B, Whang PG, Iglesias R, Wang JC, Lieberman JR. Osteoinductivity of commercially available dematerialized bone matrix: Preparations in a spine fusion model. J Bone Joint Surg Am. 2004;86(10):2243-2250.
58. Wang JC, Alanay A, Mark D, et al. A comparison of commercially available demineralized bone matrix for spinal fusion. Eur Spine J. 2007;16(8):1233-1240.
59. Kang J, An H, Hilibrand A, Yoon ST, KavanaghE, Boden S. Grafton and local bone has comparable outcomes to iliac crest bone in instrumented single-level lumbar fusions. Spine. 2012;37(12):1083-1091.
60. Kado KE, Gambetta LA, Perlman MD. Uses of Grafton for reconstructive foot and ankle surgery. J Foot Ankle Surg. 1996;35(1):59 –66.
61. Michelson JD, Curl LA. Use of demineralized bone matrix in hindfoot arthrodesis. Clin Orthop. 1996;325:203–208.
62. Stevenson S. Biology of bone grafts. Orthop Clin North Am. 1999;30(4):543–552.
63. Thordarson DB, Kuehn S. Use of demineralized bone matrix in ankle/hindfoot fusion. Foot Ankle Int. 2003;24(7):557–560.
64. Marx RE, Carlson ER, Eichstaedt RM, et al. Platelet-rich plasma: growth factor enhancement for bone grafts. Oral Surg Oral Med Oral Pathol Oral Radiol Endod. 1998;85(6):638-46.
65. Pietrzak WS, Miller SD, Kucharzyk DW, Lessek TP, Hamman NM, Woodell J. Demineralized bone graft formulations: design, development, and a novel example. Proceedings of the Pittsburgh Bone Symposium, Sheraton Station Square Hotel, Pittsburgh, PA, August 20-23, 2003;557-576.