INTRODUCTION
Most operatively and nonoperatively managed fractures heal, but a considerable number fail to unite. This subset of fractures with nonunion continue to present a treatment challenge for orthopaedic surgeons. Causes that contribute to the formation of nonunion include biological, mechanical, patient, and injury factors, and frequently the cause of nonunion may be multifactorial. Successful management can often require that multiple factors are addressed concurrently. Common difficulties encountered in the treatment of nonunions include managing infection, addressing impaired biology, and assessing patients for metabolic disorders, which compromise their healing capacity. Successful management of nonunions requires a systematic approach to identifying and addressing these issues, in addition to addressing the mechanical environment.
MANAGING INFECTION IN THE SETTING OF NONUNION
Infection is an important consideration in the workup and treatment of a patient with nonunion. When assessing for infection, consideration should be given to risk factors of infection, including patient factors such as conditions of immune compromise, malnutrition, or smoking status and injury factors such as open fractures, delayed wound healing, or previous external fixation. Infection should be strongly considered as a potential contributing factor to nonunion in the presence of any of these risk factors.1 In addition, any patient presenting with a fracture nonunion after surgical treatment should be considered and worked up for infection.
Stucken et al reported on a large series of patients with nonunion who were considered at risk of infection based on the risk factors described above.2 They reported on a cohort of 93 patients who underwent a standardized preoperative and intraoperative workup for infection, including standardized blood work [white blood cell (WBC) count, erythrocyte sedimentation rate], and C-reactive protein, nuclear studies (a combined WBC/sulfur colloid scan), and intraoperative Gram staining, and WBC count per high powered field. The authors reported that the use of simple blood tests (WBC count, erythrocyte sedimentation rate, and C-reactive protein) provided the best predictors of infection, particularly when the results of those 3 tests were used in combination. Their recommendation was that these simple blood tests alone be used for the preoperative assessment of infection.
Intraoperative cultures are the gold standard for the diagnosis of infection and should be obtained from any patient undergoing revision surgery for nonunion. Olszewski et al reported a multicenter series of a large cohort of patients undergoing revision surgery for nonunion who had a negative workup for infection (no clinical signs of infection and negative blood work) but were considered at risk because of the presence of risk factors.1 Four-hundred and fifty-three at-risk patients had intraoperative cultures sent at the time of revision surgery and 91 patients (20%) had a “surprise” positive culture. The majority (>90%) were treated with culture-specific antibiotics, whereas a small percentage (9%) of results were regarded as contaminants. Most cultures grew coagulase-negative staphylococci. Overall, the results demonstrated that those patients who had a “surprise” positive culture had lower union rates (73% vs. 96%), a higher chance of recurrent infection (12% vs. 4%), and required more secondary surgeries than those who had a negative culture. However, successful treatment was achieved in 92% of those patients with a “surprise” positive culture who received antibiotic treatment, with 21% of those requiring additional surgery to achieve union or eradicate infection.
When the diagnosis of infection is confirmed before treatment in the setting of nonunion, a staged treatment approach is recommended. The initial stage should consist of debridement, removal of any loose or chronically infected hardware, revision fixation/stabilization of the nonunion, and the treatment of infection with culture-specific local and systemic antibiotics. Local antibiotic treatment can be achieved by a variety of methods including antibiotic nails,3 antibiotic cement beads,4 bioresorbable antibiotic pellets,4 or antibiotic cement spacers. Antibiotic cement spacers can not only help to eradicate infection but can also promote the formation of an osteogenic induced membrane (Masquelet technique).5 There are also a number of options for revision fixation including both temporary (external fixation and antibiotic nails) and definitive (intramedullary nails and plates) forms of fixation. Soft tissue coverage may also be required in the form of flap or rotational coverage at this stage as well. The second stage generally proceeds after a period of systemic antibiotic therapy and when both clinical and serological signs of infection are absent. This reconstructive stage to address the nonunion may consist of definitive fixation, bone grafting, other biologic treatment, or bone transport, depending on the specific characteristics of the nonunion.6,7
BONE GRAFTING: WHAT IS THE IDEAL TYPE?
The mainstay of surgical treatment for nonunions with impaired biology (atrophic nonunion) is autologous bone grafting. Three attributes are essential for successful grafting: osteogenesis, osteoinduction, and osteoconduction. Osteogenesis is defined as a cell's ability to differentiate into a bone matrix producing osteoblasts. Osteoinduction is the ability of a growth factor to induce osteogenic differentiation of skeletal progenitor cells or induce osteoid deposition by osteoblasts. Osteoconduction describes the ability of a material to provide a scaffold for the attachment and subsequent bone matrix deposition of bone-forming cells. All 3 elements are essential for successful healing in the setting of atrophic nonunion.8
Autograft has been the gold standard for atrophic nonunion treatment for decades.9 Bone graft, harvested from any location within the skeleton, possesses all 3 of the above-mentioned qualities. In addition, its risk profile with regard to disease transmission and surgical complications is favorable when compared with allograft and recombinant growth factor use. Last, acquisition of autograft is cost-effective, particularly when compared with other off-the-shelf products. However, there are several disadvantages that exist with autograft use. The most commonly cited complication is harvest site pain, although recent studies have reported that with precise surgical technique, the use of local anesthesia and appropriate postoperative analgesia, these sequelae can be minimized.10 Costs associated with increased operating room time and length of stay have also been cited as a possible limitation of autograft.11
The 2 most common options for autograft harvest include iliac crest bone graft (ICBG) and the Reamer-Irrigator-Aspirator (RIA). Both options provide large quantities of bone graft. RIA can harvest up to 60 ccs of graft material, whereas the ICBG can provide approximately 30 ccs. Bone graft harvested with both methods contains skeletal progenitor cells, osteoblasts, monocytes, and bone lining cells, and progenitor cells within the harvested graft material have been shown to be capable of tripotent differentiation (able to form bone, cartilage, and fat).12 A commonly cited potential disadvantage of RIA graft harvest is the relatively low number of bone-forming cells present within the graft material because these cells are flushed away through the collection filter due to the high flow rate of the irrigation fluid.13 A randomized controlled trial comparing the 2 harvest options revealed that the RIA system was overall more cost-effective than posterior ICBG because of the longer operating room time associated with the graft harvest from the posterior superior iliac spine.14 There was no difference in the union rate between RIA (82%) and ICBG (86%) and no difference in the complication rate, although graft harvest site pain was reported to be lower with the RIA.
Although autograft does provide the necessary biologic elements, recent trends have indicated a shift toward other forms of bone healing augmentation, often less painful, less time-consuming, and less invasive alternatives. The question arises whether it is time for a new gold standard for the treatment of nonunions.
Although the young skeleton is rich in skeletal progenitor cells, this abundance of bone-forming cells decreases with age, and recent research has shown that the frequency of bone progenitor cells in the middle aged and elderly is significantly reduced.15 Bone marrow aspirate concentrate (BMAC), obtained by bone marrow aspiration and centrifugation, is currently used to increase the nucleated cell fraction of bone marrow aspirate and by doing so increases the number of bone progenitor cells within a certain volume. Therefore, BMAC may represent a superior option to ICBG or RIA because of the increased number of osteogenic cells, which directly contribute to the bone-forming potential of the graft, particularly in the middle-aged and elderly patients. Both autograft and exogenous sources of osteoinductive proteins [such as bone morphogenetic proteins (BMPs) and platelet-derived growth factor] supply pro-osteogenic cues to the site of desired bone healing.16 However, both do so in supraphysiological doses and without adhering to any physiologic timeline for release. By contrast, BMAC provides a concentrated population of cells, which release physiologic doses of osteogenic growth factors, in a timed, coordinated manner, which is responsive to the microenvironment. Therefore, BMAC may represent the best-suited substitute for osteoinductive agents. Finally, decades of research has provided a plethora of commercially available osteoconductive scaffold options with varying pore sizes, resorption rates, and mechanical/handling properties, and the osteoconductive properties of autograft are easily replaced with unlimited, off-the-shelf supply. Therefore, BMAC in combination with commercially available, osteoconductive scaffolds may provide the optimal combination of osteogenic cells, osteoinductive proteins, and limitless graft volume, without the invasive techniques required to harvest autograft and thus may represent the approach of the future.
BIOLOGIC THERAPIES FOR NONUNION
Many biological and mechanical factors have been implicated in the etiology of fracture nonunion, and frequently more than 1 factor is present. When abundant callus formation is present, such as in a hypertrophic nonunion, typically improved mechanical stabilization with internal fixation and possibly compression is effective to achieve healing. However, in the setting of atrophic nonunion, where minimal callus formation is present, appropriate treatment includes augmentation of the biologic potential at the nonunion site. Traditionally, autologous ICBG has been the gold standard for this purpose. However, concerns over donor site morbidity have led surgeons to seek alternative sources of osteogenesis. More recently, intramedullary femoral reaming and debris aspiration (RIA bone graft) has provided a promising source of osteogenic material, however this still requires a separate surgical procedure and has its own unique complication profile.17,18 In addition, both iliac crest autograft and RIA graft have limited supply. Investigators continue the search for an effective biologic material to augment nonunion healing that avoids the complications associated with harvest and is not limited in its quantity. Several options are currently available including demineralized bone matrix (DBM), BMPs, and systemic parathyroid hormone (PTH) therapy.
In 1965, Marshall Urist identified a substance in demineralized bone that led to osteoinduction in extraskeletal sites (heterotopic bone formation).19 Acid extraction of allogenic bone leaves a residual of type I collagen, other noncollagenous proteins, and multiple growth factors. This growth factor profile was subsequently identified as the transforming growth factor β superfamily. Among these, BMPs comprised at least 15 of the proteins responsible for osteogenesis. The extraction process was streamlined and commercialized, and DBM was approved by the FDA. Since that time, several methodologically weak studies have suggested clinical efficacy of DBM for nonunion treatment.20,21 At present, the variability in growth factor concentration between products and between lots of a single product,22 as well as lack of Level I evidence regarding clinical efficacy, has limited its widespread use for nonunion treatment.
Among the transforming growth factor β proteins, several BMPs were identified to be potently osteoinductive. BMP-2 and BMP-7 were commercialized. BMP-7 was FDA approved for recalcitrant nonunions based on the pivotal study by Friedlaender et al.23 In this study, 124 tibial nonunions were treated with reamed intramedullary nailing and randomized to autograft or BMP-7. Equivalent union and complication rates were reported in the 2 treatment groups, and the authors concluded that BMP-7 was a safe and effective alternative to autograft in this setting. Since that time, reports of complications in spine surgery, questions about appropriate dose, the formation of antibodies, and the high cost of this compound have decreased initial enthusiasm.
Subcutaneous intermittent injection of PTH is potently osteogenic, and has recently been commercialized as teriparatide (Forteo, Eli Lilly). This agent is FDA approved and very effective in osteoporosis treatment. Animal studies have been conclusive in showing its strong efficacy in stimulating bone formation in fracture healing and fusions.24 Several human studies have been conducted in acute fractures,25,26 and although early evidence is promising, clear indications are lacking. This systemic therapy for achieving nonunion healing is currently limited to case reports but may be a useful adjunct in the future.
The search for the ideal biologic stimulant in nonunion treatment persists. Autografts, both from the iliac crest and the femoral canal, provide reliable substrates but are limited by complications and quantity. Novel approaches and materials continue to be developed and tested. Before selecting an autograft alternative, the surgeon should become familiar with the advantages and disadvantages of that product and should critically analyze the evidence supporting its use.
CELL-BASED THERAPIES FOR NONUNION MANAGEMENT
Few recent developments in orthopaedic basic science garner more interest than cell-based therapies for bone regeneration. However, compared with the other critical components of bone formation, surgeons have had the most limited access to a bone-forming cell population. A myriad of alternatives are currently available to surgeons at the point of care, although high-quality clinical data remain elusive. Three of the most actively studied technologies/techniques are viable allograft products, lipoaspirate cell harvest, and endosteal bone harvest via reaming aspiration.
Viable allografts represent an interesting product concept available to surgeons at the point of care as a ready-to-use, packaged product. A limited number of these products are available at this time. Conceptually, the development of these products followed improvements in the cadaver donor screening process, which improved the safety and comfort level with the use of fresh allograft material. These products required the development of proprietary techniques to harvest viable mesenchymal stem cells from fresh allograft bone tissue. In these products, the cells are then provided in some form of carrier/vehicle, such as DBM and/or allograft chips as a complete, free-standing regenerative matrix. The distinguishing feature of this type of complete graft substitute concept relies on the presence of a viable cell population. One study of a specific viable allograft product provided a functional and comparative analysis.27 The authors demonstrated functionally competent mesenchymal cells and cell numbers similar to a typical BMAC procedure. Clinical reports of the viable allograft combinations are limited but include studies of the spine, foot and ankle, and maxillofacial reconstructions.28,29 All of these studies are level 4 case series, and high-level evidence regarding the efficacy of viable allografts is lacking. However, there are no reports of disease transmission to date, and their early clinical safety profile seems favorable. Further studies are necessary to understand their efficacy and to clarify indications.
Lipoaspirates are a newer concept for autologous mesenchymal cell harvest. This technique represents a simple, yet elegant approach for physical separation of cells from a manual lipoaspirate and uses a proprietary canister to isolate the desired cell population. This technique was developed to obtain microfragmented adipose tissue with an intact stromal vascular niche and mesenchymal cells with a high regenerative capacity.30 This technique represents the newest and most unique approach to cell harvest for bone regeneration, and clinical experience with its use has been very limited. A proof-of-concept study demonstrates one of the proposed techniques for autologous cell regeneration including ex vivo cell expansion, optimization of delivery using DBM, followed by second-stage reimplantation.31 Of the 3 patients reported in the article, 1 patient healed, 1 developed nonunion, and 1 experienced a deep infection. Clearly further basic science and clinical studies are required to better understand the osteogenic potential of this cell population and evaluate its role in clinical applications.
Intramedullary bone graft harvest (RIA) is likely the most studied technique for accessing autogenous cells for regenerative applications. Several publications have evaluated intramedullary reaming debris as a source of viable osteoblastic progenitor cells.32,33 A large number of cells are found on the surface of the bony spicules liberated during the mechanical reaming process. Two further considerations warrant a further analysis of the possibility that endosteal reaming with RIA may represent an optimal approach to liberating a potent bone progenitor cell population. First, there is a traditional schema with an osteoblastic cell niche along the endosteal surface with osteoblasts and hematopoietic precursors and a separate vascular niche more centrally along sinusoids with the mesenchymal cell population. A recent study used a novel technique to enzymatically separate cells from the central and endosteal regions.34 A significant number of progenitors were found in both regions. In fact more progenitors were found along the endosteum, and these cells had a higher proliferative capacity. This finding suggests that mechanical reaming of the endosteal zone likely liberates a large number of mesenchymal progenitor cells.
Further support for this cell harvesting concept comes from evaluations of the waste water from the reaming process, with the RIA. One study compared the progenitor cell numbers in samples of RIA waste fluid from patients undergoing nonunion treatment with age-matched bone marrow aspirate samples in trauma patients.35 All of the RIA samples had cell counts well above the classically referenced threshold for successful healing from Hernigou et al of 55,00036; in fact, the average number of cells was over 300,000, representing a very high volume of liberated progenitors. Further studies are underway to improve the ability to concentrate progenitors liberated from the RIA at the point of care. Theoretically, surgeons will soon have the ability to enrich structural and inductive grafts of choice with a large number of endogenous bone-forming progenitors.
METABOLIC WORKUP OF NONUNIONS
Impaired fracture healing occurs in approximately 5%–10% of all patients, and a subset of these patients will develop a fracture nonunion, where bony union will not occur without further intervention. The etiology of a nonunion can be multifactorial and include characteristics of the fracture, such as mechanical instability, insufficient vascularity, and poor surface characteristics and suboptimal bony contact.37 In addition, patient factors including cigarette smoking, alcohol abuse, malnutrition, and anti-inflammatory medication use can be detrimental to fracture healing.38–42 However, some fractures fail to unite despite excellent fixation and a seemingly heathy host with adequate local biology. In this subset of patients who present with a fracture nonunion, metabolic and endocrine abnormalities should be suspected as a potential etiology for their nonunion.
Several metabolic abnormalities have been associated with impaired fracture healing including vitamin D deficiency, diabetes, hypogonadism, and imbalances of calcium, growth hormone, and PTH.37,38,43 Of these, disorders affecting vitamin D, calcium, and PTH most directly affect bone metabolism during fracture healing and have a prevalence of up to 50% in certain populations.37,44,45
In patients with a nonunion, metabolic bone disease should be suspected when one or more of the following criteria are present: (1) a persistent nonunion despite adequate treatment without obvious technical error, (2) a history of multiple low-energy fractures with at least 1 progressing to a nonunion, and (3) a nonunion of a minimally displaced pubic rami or sacral ala fracture. When these criteria were used, 84% of patients with a nonunion were newly diagnosed with one or more metabolic or endocrine abnormalities, including vitamin D deficiency, calcium imbalances, central hypogonadism, thyroid disorders, and PTH disorders.43
Patients identified using these criteria should undergo metabolic and endocrine evaluation, with blood and urine laboratory testing for abnormalities in a defined set of vitamins, minerals, and hormones related to bone healing (Table 1). In addition to vitamin D metabolites, PTH, calcium, and phosphate, the laboratory testing should also include stress-related hormones (adrenocorticotropic hormone and cortisol), anabolic (dehydroepiandrosterone sulfate and growth hormone) and sex (follicle stimulating hormone, estrogen, estradiol, and testosterone) hormones, thyroid function tests, and alkaline phosphatase, a marker of bone turnover. A comprehensive endocrine and metabolic profile is used rather than a few specific metabolic tests so that any imbalances can be interpreted in the context of a full profile.
;)
TABLE 1.: Metabolic and Endocrine Related Laboratory Tests (Serum Tests Unless Otherwise Noted)
Once a patient with a nonunion has been diagnosed with an endocrine or metabolic disorder, treatment should be performed using a team-based approach. Medical management of all diagnosed endocrine and metabolic disorders should be performed by an endocrinologist with specialization or experience in metabolic bone disease. Management of the nonunion with operative and nonoperative methods should be performed by an orthopaedic surgeon. In general, medical treatment should not inhibit surgical intervention. However, medical management alone may induce nonunion healing without surgery by providing a biologic stimulus. In a group of 31 patients with well-established nonunions and a newly diagnosed metabolic disorder, 8 patients were healed of their nonunion following medical treatment alone without operative intervention,43 highlighting the importance of these metabolic and endocrine abnormalities for the nonunion etiology.
CONCLUSIONS
The etiology of nonunion is frequently multifactorial, and often successful treatment requires the assessment and management of multiple factors. Infection, metabolic disorders, and impaired biology at the fracture site are important considerations in addition to the mechanical environment. An understanding of the variety of available options for treating these and the evidence supporting their use is critical to success in nonunion management.
REFERENCES
1. Olszewski D, Streubel PN, Stucken C, et al. Fate of patients with a “surprise” positive culture after
nonunion surgery. J Orthop Trauma. 2016;30:e19–e23.
2. Stucken C, Olszewski DC, Creevy WR, et al. Preoperative diagnosis of
infection in patients with nonunions. J Bone Joint Surg Am. 2013;95:1409–1412.
3. Selhi HS, Mahindra P, Yamin M, et al. Outcome in patients with an infected
nonunion of the long bones treated with a reinforced antibiotic bone cement rod. J Orthop Trauma. 2012;26:184–188.
4. McKee MD, Li-Bland EA, Wild LM, et al. A prospective, randomized clinical trial comparing an antibiotic-impregnated bioabsorbable bone substitute with standard antibiotic-impregnated cement beads in the treatment of chronic osteomyelitis and infected
nonunion. J Orthop Trauma. 2010;24:483–490.
5. Stafford PR, Norris BL. Reamer-irrigator-aspirator
bone graft and bi Masquelet technique for segmental bone defect nonunions: a review of 25 cases. Injury. 2010;41(suppl 2):S72–S77.
6. Sadek AF, Laklok MA, Fouly EH, et al. Two stage reconstruction versus bone transport in management of resistant infected tibial diaphyseal
nonunion with a gap. Arch Orthop Trauma Surg. 2016;136:1233–1241.
7. Nauth A, McKee MD, Einhorn TA, et al. Managing bone defects. J Orthop Trauma. 2011;25:462–466.
8. Khan SN, Cammisa FP Jr, Sandhu HS, et al. The biology of bone grafting. J Am Acad Orthop Surg. 2005;13:77–86.
9. Sen MK, Miclau T. Autologous iliac crest
bone graft: should it still be the gold standard for treating nonunions? Injury. 2007;38(suppl 1):S75–S80.
10. Loeffler BJ, Kellam JF, Sims SH, et al. Prospective observational study of donor-site morbidity following anterior iliac crest bone-grafting in orthopaedic trauma reconstruction patients. J Bone Joint Surg Am. 2012;94:1649–1654.
11. Ackerman SJ, Mafilios MS, Polly DW Jr. Economic evaluation of bone morphogenetic protein versus autogenous iliac crest
bone graft in single-level anterior lumbar fusion: an evidence-based modeling approach. Spine (Phila Pa 1976). 2002;27(16 suppl 1):S94–S99.
12. Kuehlfluck P, Moghaddam A, Helbig L, et al. RIA fractions contain mesenchymal stroma cells with high osteogenic potency. Injury. 2015;46(suppl 8):S23–S32.
13. Sagi HC, Young ML, Gerstenfeld L, et al. Qualitative and quantitative differences between
bone graft obtained from the medullary canal (with a Reamer/Irrigator/Aspirator) and the iliac crest of the same patient. J Bone Joint Surg Am. 2012;94:2128–2135.
14. Dawson J, Kiner D, Gardner W II, et al. The reamer-irrigator-aspirator as a device for harvesting
bone graft compared with iliac crest
bone graft: union rates and complications. J Orthop Trauma. 2014;28:584–590.
15. Zhou S, Greenberger JS, Epperly MW, et al. Age-related intrinsic changes in human bone-marrow-derived mesenchymal
stem cells and their differentiation to osteoblasts. Aging Cell. 2008;7:335–343.
16. Nauth A, Giannoudis PV, Einhorn TA, et al. Growth factors: beyond bone morphogenetic proteins. J Orthop Trauma. 2010;24:543–546.
17. Calori GM, Colombo M, Mazza EL, et al. Incidence of donor site morbidity following harvesting from iliac crest or RIA graft. Injury. 2014;45(suppl 6):S116–S120.
18. Lowe JA, Della Rocca GJ, Murtha Y, et al. Complications associated with negative pressure reaming for harvesting autologous
bone graft: a case series. J Orthop Trauma. 2010;24:46–52.
19. Urist MR. Bone: formation by autoinduction. Science. 1965;150:893–899.
20. Pieske O, Wittmann A, Zaspel J, et al. Autologous
bone graft versus demineralized bone matrix in internal fixation of ununited long bones. J Trauma Manag Outcomes. 2009;3:11.
21. Wilkins RM, Kelly CM. The effect of allomatrix injectable putty on the outcome of long bone applications. Orthopedics. 2003;26(suppl 5):s567–s570.
22. Bae HW, Zhao L, Kanim LE, et al. Intervariability and intravariability of bone morphogenetic proteins in commercially available demineralized bone matrix products. Spine (Phila Pa 1976). 2006;31:1299–1306; discussion 1307–1298.
23. Friedlaender GE, Perry CR, Cole JD, 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):S151–S158.
24. Zhang D, Potty A, Vyas P, et al. The role of recombinant PTH in human
fracture healing: a systematic review. J Orthop Trauma. 2014;28:57–62.
25. Aspenberg P, Genant HK, Johansson T, et al. Teriparatide for acceleration of fracture repair in humans: a prospective, randomized, double-blind study of 102 postmenopausal women with distal radial fractures. J Bone Miner Res. 2010;25:404–414.
26. Peichl P, Holzer LA, Maier R, et al. Parathyroid hormone 1-84 accelerates fracture-healing in pubic bones of elderly osteoporotic women. J Bone Joint Surg Am. 2011;93:1583–1587.
27. Baboolal TG, Boxall SA, El-Sherbiny YM, et al. Multipotential stromal cell abundance in cellular bone allograft: comparison with fresh age-matched iliac crest bone and bone marrow aspirate. Regen Med. 2014;9:593–607.
28. Eastlack RK, Garfin SR, Brown CR, et al. Osteocel Plus cellular allograft in anterior cervical discectomy and fusion: evaluation of clinical and radiographic outcomes from a prospective multicenter study. Spine (Phila Pa 1976). 2014;39:E1331–E1337.
29. Scott RT, Hyer CF. Role of cellular allograft containing mesenchymal
stem cells in high-risk foot and ankle reconstructions. J Foot Ankle Surg. 2013;52:32–35.
30. Tremolada C, Colombo V, Ventura C. Adipose tissue and mesenchymal
stem cells: state of the art and lipogems(R) technology development. Curr Stem Cell Rep. 2016;2:304–312.
31. Dufrane D, Docquier PL, Delloye C, et al. Scaffold-free three-dimensional graft from autologous adipose-derived
stem cells for large bone defect reconstruction: clinical proof of concept. Medicine (Baltimore). 2015;94:e2220.
32. Frolke JP, Bakker FC, Patka P, et al. Reaming debris in osteotomized sheep tibiae. J Trauma. 2001;50:65–69; discussion 69–70.
33. Wenisch S, Trinkaus K, Hild A, et al. Human reaming debris: a source of multipotent
stem cells. Bone. 2005;36:74–83.
34. Siclari VA, Zhu J, Akiyama K, et al. Mesenchymal progenitors residing close to the bone surface are functionally distinct from those in the central bone marrow. Bone. 2013;53:575–586.
35. Churchman SM, Kouroupis D, Boxall SA, et al. Yield optimisation and molecular characterisation of uncultured CD271+ mesenchymal
stem cells in the Reamer Irrigator Aspirator waste bag. Eur Cell Mater. 2013;26:252–262.
36. Hernigou P, Poignard A, Beaujean F, et al. Percutaneous autologous bone-marrow grafting for nonunions. Influence of the number and concentration of progenitor cells. J Bone Joint Surg Am. 2005;87:1430–1437.
37. Brinker MR, O'Connor DP. The biological basis for nonunions. JBJS Rev. 2016;4.
38. Copuroglu C, Calori GM, Giannoudis PV. Fracture non-union: who is at risk? Injury. 2013;44:1379–1382.
39. Bergenstock M, Min W, Simon AM, et al. A comparison between the effects of acetaminophen and celecoxib on bone
fracture healing in rats. J Orthop Trauma. 2005;19:717–723.
40. Castillo RC, Bosse MJ, MacKenzie EJ, et al. Impact of smoking on
fracture healing and risk of complications in limb-threatening open tibia fractures. J Orthop Trauma. 2005;19:151–157.
41. Kayal RA, Tsatsas D, Bauer MA, et al. Diminished bone formation during diabetic
fracture healing is related to the premature resorption of cartilage associated with increased osteoclast activity. J Bone Miner Res. 2007;22:560–568.
42. Einhorn TA, Bonnarens F, Burstein AH. The contributions of dietary protein and mineral to the healing of experimental fractures. A biomechanical study. J Bone Joint Surg Am. 1986;68:1389–1395.
43. Brinker MR, O'Connor DP, Monla YT, et al. Metabolic and endocrine abnormalities in patients with nonunions. J Orthop Trauma. 2007;21:557–570.
44. Holick MF. High prevalence of vitamin D inadequacy and implications for health. Mayo Clin Proc. 2006;81:353–373.
45. Bogunovic L, Kim AD, Beamer BS, et al. Hypovitaminosis D in patients scheduled to undergo orthopaedic surgery: a single-center analysis. J Bone Joint Surg Am. 2010;92:2300–2304.
