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Scientific Understanding of the Induced Membrane Technique: Current Status and Future Directions

Yee, Michael A. MD*; Mead, Matthew P. MD; Alford, Andrea I. PhD*,‡; Hak, David J. MD, MBA, FACS§; Mauffrey, Cyril MD, FACS, FRCS§; Hake, Mark E. MD*

Journal of Orthopaedic Trauma: October 2017 - Volume 31 - Issue - p S3–S8
doi: 10.1097/BOT.0000000000000981
Supplement Article
Free

Objectives: To review the most recent basic science advances made in relation to the induced membrane technique and how those relate to clinical practice, applications, and future research directions.

Design: Review of the literature.

Setting: Any trauma center which might encounter large segmental bone defects.

Articles Reviewed: Basic science articles that looked at characteristics of the induced membrane published in the past 30 years.

Intervention: None.

*Department of Orthopaedic Surgery, University of Michigan Health System, Ann Arbor, MI;

Department of Surgery, Michigan State University, Sparrow Health System, Lansing, MI;

Department of Biomedical Engineering, University of Michigan, Ann Arbor, MI; and

§Department of Orthopaedics, Denver Health Medical Center, Denver, CO.

Reprints: Cyril Mauffrey, MD, FACS, FRCS, Department of Orthopaedics, Denver Health Medical Center, 777 Bannock St, 80204 Denver, CO (e-mail: Cyril.mauffrey@dhha.org).

Authors have no conflicts in relation to this manuscript.

Accepted July 14, 2017

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INTRODUCTION

Clinical management of large bone defects poses a significant challenge in the field of orthopaedics. Originally developed in the 1970s by AC Masquelet, the Masquelet technique is a 2-stage bone reconstruction procedure that has gained popularity over the past decade. The key points of this strategy include control of local infection with radical debridement, placement of a polymethylmethacrylate (PMMA) spacer to preserve dead space, and induction of a membrane that protects against graft resorption and promotes bony ingrowth into the defect.1 Bone union rates using the Masquelet technique range from 82.6% to 100%.2–5 This is comparable with other salvage techniques, such as Ilizarov bone transport (83%–100% union)6 and vascularized fibular autograft (88%–100% union).7–9 However, the Masquelet technique has several advantages over these methods, including decreased operating time, technical ease, and low resource utilization.10,11 In addition, the Masquelet technique is length independent, meaning that both large and small osseous defects can be treated with similar success.12,13

The purpose of this article is to review the body of basic science research investigating various modifiable aspects of the Masquelet technique. This includes work performed in both animal models and human patients. There has been considerable interest in the properties of the induced membrane (IM) and its potential role in graft osteointegration. Likewise, several authors have investigated whether differing bone grafts or bone graft supplements improved clinical outcomes. It is our hope that providing an outline of the current state of research within this field may provide guidance to direct further research into the most clinically meaningful areas.

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BRIEF DESCRIPTION OF THE PROCESS OF THE MASQUELET TECHNIQUE

Several authors have now published detailed tutorials on how to perform the Masquelet technique.1,2,11,12,14–17 Over time, some aspects of the procedure have been modified such as the addition of antibiotics into the PMMA spacer for local antibiotic elution,18 or preferential use of intramedullary nailing instead of external fixation.14,15 Nevertheless, the core components of the original procedure remain unchanged and can be found in the above-referenced articles.

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HISTOLOGIC AND MOLECULAR CHARACTERISTICS OF THE IM

The histologic characteristics of the IM have been described in multiple studies.19–27 Initially, there is an inflammatory reaction to the PMMA spacer with inflammatory cell infiltration and edema, contributing to the formation of the IM.19,28 As early as 2 weeks after PMMA implantation, the IM demonstrates organization into inner and outer components. The inner component (approximating the PMMA spacer) consists of densely packed, cuboidal,23 or round22,26 fibroblast-like cells. The outer component is made of a combination of fibroblasts and myofibroblasts in a type-I collagen matrix.19,20,25,27 Initially, the collagen fibers and fibroblasts are oriented parallel to the PMMA spacer, but with increasing distance from the cement interface, they become more random in their orientation and eventually blend with surrounding tissues.22–24 At 3 weeks, 3 distinct histological “zones” can be identified in the outer layer. As described by Gouron et al, zone A consists of the thin, highly cellular layer in contact with the PMMA spacer. Zone B consists of a network of collagen fibers oriented parallel to the PMMA spacer, which increases in size with time. Zone C is the thickest and outermost layer of the IM and consists of loose, disorganized tissue with a large vascular network.24

Although Cuthbert et al29 proposed changing the nomenclature from “induced membrane” to “induced periosteum,” significant differences have been observed between the IM and periosteum. Although both the periosteum and IM are arranged in inner and outer layers, there is a sharp demarcation between layers in the periosteum compared with a gradual transition from the inner to outer layer based on the orientation of fibroblasts in the IM. Furthermore, the IM is thicker, more cellular, and more vascular than the periosteum. Although concentrations of PGE-2 and BMP-2 are similar between the 2, the periosteum contains higher concentrations of vascular endothelial growth factor (VEGF), FGF-2, and ANG-II than the IM.28

Vascularity is seen predominately in the outer portions of the membrane, whereas areas closest to the PMMA spacer are least vascular. Of importance, membrane vascularization increases significantly over time, with the greatest increase occurring between 2 and 4 weeks.22 By 4 weeks, an increase in larger caliber blood vessels can be seen.19,23,28 After 4–6 weeks, there is a progressive decrease in vascularity, which has been shown in both animal and human models.22,27,28 Samples taken from membranes formed around human tibial and femoral defects at 3 months have <60% of the vascularity of 1-month-old membranes.27

Inflammatory cell changes occur along a similar time course to that of membrane vascularity. Neutrophils and eosinophils can be seen concentrated around blood vessels and the PMMA surface,23 consistent with an inflammatory reaction to the PMMA spacer. A rat model shows that this inflammatory reaction completely resolves by 6–8 weeks.19 Similarly, in a sheep model, there was a lack of inflammatory reaction (by histology as well as immunohistochemical staining for CD14+ cells) at 6 weeks.20

Several studies have demonstrated that the IM is a source of mesenchymal stem cells (MSCs).20,22,26,28–32 CD271-positive cells, a marker of bone marrow–derived MSCs, have been noted in high abundance in human IMs.29 Both human and animal models show that IM-derived MSCs are multipotent, with the capability to transform into adipogenic, osteogenic, and chondrogenic lineages.30–32 Gruber et al32 noted evidence of osteogenic differentiation of MSCs in human IM's over a broad range of times, from as early as 6 weeks (youngest IM sampled) to as late as 17 weeks. In a rat model, Patel et al30 demonstrated that a subset of cells within the IM expressed markers of both embryonic and adult stem cells and are shown to be of mesenchymal origin rather than hematopoietic lineage. Henrich et al22 and Nau et al26 used rat femoral defect models to identify MSCs from harvested membranes over time, finding that MSCs were present at 2 weeks after PMMA spacer placement, but disappeared at weeks 4 and 6.

As discussed, MSCs can differentiate down osteogenic lineages, and studies conducted on sheep, rat, and human models have confirmed the presence of osteoblasts within the IM.20,24,32,33 Furthermore, multiple authors make note of some immature and mature bone and cartilage foci that can occur within24,31–33 or external to the IM20 before grafting. In a human study (n = 12), the presence or absence of intramembranous bone formation did not correlate with spacer duration.32 In addition, human studies have shown that the primitive vascular tissue located within the IM has the capability to differentiate toward calcified tissue, in some cases even mature lamellar bone.27

In addition to identifying osteoblasts within the IM, considerable work has been conducted to characterize changes in angiogenic and osteogenic protein secretion by the IM over time.19,22,23,28,33 In a rabbit model, levels of VEGF released by the IM were highest at week 2 and remained high (with slight downward trend) until 8 weeks.19 VEGF promotes angiogenesis at injury sites and participates in the mobilization of MSCs in bone marrow.28,34 The production of BMP-2, an initial signaling molecule in osteoblast differentiation, peaked at 4 weeks, followed by a decline in week 6, and return to baseline levels at week 8.24 Similarly, Henrich et al22 found that the BMP-2 protein content increased significantly from 2 to 4 weeks, but remained unchanged at 6 weeks in a rat model. TGFβ remained elevated at all time points. Christou et al23 reported similar findings of VEGF and BMP-2 staining located throughout the IM, but most concentrated near the PMMA spacer (zone A) in an ovine model. Wang et al28 expanded on these findings in a rabbit model, reporting that VEGF and BMP-2 expression peaked at 6 weeks and then began to decline, whereas ANG-II, a promoter of angiogenesis, peaked at week 4 and then began to decline. In humans, Aho et al27 showed that VEGF expression decreases drastically in membranes after 1 month, which correlated with histologic analyses that showed decreasing vascularity as membranes became more mature over time. They also noted that IL-6 and type-I collagen expression remained highest in early membranes (<1 month old), whereas expression was minimal in membranes >2 months old. Furthermore, membranes harvested 1 month after PMMA spacer insertion induced osteoblastic MSC differentiation at a greater rate than membranes harvested at 2 months after PMMA spacer insertion. Thus, the results of these animal and human studies suggest that optimal biochemical conditions for stage 2 of the Masquelet technique are between 4 and 6 weeks. As previously discussed, the 4–6-week period is also when the membrane is most vascular, further supporting this to be the optimal biochemical time course for stage 2 grafting. However, adequate membrane thickness of 1–2 millimeters required for surgical handling during spacer removal is often not achieved until 6–8 weeks.11

The osseous defect environment may also have an influence on the expression of angiogenic and osteogenic proteins. Henrich et al22 compared characteristics of membranes induced at subcutaneous sites versus femoral defect sites (thus possessing a muscular envelope) in a rat model. They found that membranes at 4 and 6 weeks after PMMA implantation had significantly higher cell number and vascularity if induced around femoral defects rather than subcutaneously. BMP-2 and TGFβ contents were also significantly higher in IMs at both 2 and 4 weeks from femoral defect sites versus subcutaneous sites. A general increase in VEGF staining is also seen in membranes induced from femoral defects as opposed to subcutaneous pockets. Furthermore, no MSCs were seen at any time point (2, 4, or 6 weeks) in IMs formed in subcutaneous sites. Although these findings have yet to be confirmed in human studies, they suggest that bone defects contained in a muscular environment (eg, femoral, humeral) may induce a more favorable membrane for healing than subcutaneous bone defects (eg, tibial and distal ulnar).

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GRAFT AND SPACER MATERIALS

Although the unique characteristics of the IM play a major part in the success of the Masquelet technique, the features of autologous bone graft or synthetic graft placed within the IM are also important. The anterior tibial metaphysis, femoral condyle, and iliac crests are conventional harvest sites for bone autograft.35 In 2000, Masquelet et al1 reported a case series of 35 long bone reconstructions using their described IM technique. Defects in this series ranged from 5 to 24 cm, and all defects were filled with cancellous iliac crest bone graft (ICBG). The reported union rate from this series was 100% (although 4/35 required a second grafting procedure to achieve union). Similarly, Risteniemi et al13 reported a 96% union rate using ICBG, although nearly a third of successful cases required secondary operations to achieve union. In the largest case series to date reporting on outcomes of the IM technique, Karger et al36 found a 90% union rate using cancellous or corticocancellous autograft. However, the specific donor site was not reported for each case, and an average of 6.11 surgical procedures was required to achieve union.

More recent innovations in the Reamer–Irrigator–Aspirator (RIA) device has afforded some advantages over conventional bone grafting, especially the ability to obtain large volume grafts containing osteoinductive and osteoconductive growth factors, as well as osteogenic MSCs that are comparable, if not superior, with the material harvested from more conventional sites.37–45 Wenisch et al39 identified multipotent stem cells within the RIA aspirate capable of differentiating along osteogenic pathways in humans. Henrich et al46 later demonstrated that RIA aspirate contained MSCs capable of higher calcium deposition than those MSCs obtained by the traditional aspiration technique from the iliac crest. Schmidmaier et al40 found higher concentrations of TGFβ, insulin-like growth factor–I, FGF-1, platelet-derived growth factor, and BMP-2 in the RIA graft when compared with ICBG, whereas concentrations of VEGF and basic fibroblast growth factor were significantly lower. Henrich et al47 propose that these differences in growth factors and osteogenic activity of MSCs may be more influenced by harvest procedure than by the tissue site itself. They suggested that the altered gene expression and function of femur-derived MSCs obtained by RIA could be due to the harsher conditions (eg, heat, pressure, and vacuum filtration) of the RIA procedure.

Several case series report on clinical outcomes among defects grafted with RIA-derived bone graft. Stafford et al4 report an average harvest volume of 47 cm3 using RIA to treat 27 tibial and femoral bone defect nonunions. The union rate at 1 year was 90% without any RIA-related complications. McCall et al48 achieved 85% union among 20 long bone defects filled with RIA-derived bone graft. They suggest that their higher rate of early failures could be attributed to over-packing the defect with graft as evaluation at revision surgery revealed that highly packed areas had not consolidated. Recently, Taylor et al2 reported an 82.6% union rate among 69 traumatic bone defects treated with the IM technique using either RIA graft or ICBG. There was no significant difference between union rates based on the type of autologous bone graft used. However, they found that deep infection, both pre- and post-grafting, was a statistically significant risk factor for nonunion, regardless of the graft material used.

Some surgeons have added BMP at the second stage of treatment in attempts to improve bone healing. Masquelet and Begue12 reported on 11 long bone defects that received BMP-7 along with ICBG, resulting in 10 successful unions and 1 nonunion. They concluded that BMP-7 did not improve outcomes in this series, but this assertion was based on historical controls, as no ICBG-only controls were included. Donegan et al49 reported on 11 cases of segmental bone defects treated with a variety of graft materials, including ICBG, RIA, cancellous allograft, demineralized bone matrix, BMP-2, and platelet-rich concentrate. BMP-2 was used in all but 2 cases, and 10 of 11 defects achieved successful union. Of interest, 5 cases of successful union were treated without any autologous bone graft, but rather with BMP-2 and a combination of allograft, demineralized bone matrix, and platelet-rich concentrate. The only case of nonunion was complicated by infection after a second stage using only ICBG to fill the defect. In a case series of 15 open supracondylar femur fractures with critical bone loss treated with the Masquelet technique using cancellous bone autograft augmented with BMP-2 or BMP-7, Dugan et al3 reported 100% union without any cases complicated by deep infection or malunion. In this series, average time to second stage procedure was 3.6 months. Although these clinical case series document successful use of growth factors and alternative graft materials, they were not designed to address possible advantages over autograft alone.

To date, there is limited success about the development of synthetic bone grafts to fill osseous defects treated with the IM technique. Viateau et al50 tested a new bioengineered coral granule bone graft substitute that was impregnated with autologous MSCs in a sheep osseous defect model 6 weeks after the placement of a PMMA spacer. Radiographs, histology, and CT performed 6 months later showed no significant differences between the amount of newly formed bone in defects filled with coral/MSCs and those filled with autograft, yet radiological scores differed significantly between the 2 groups (21% and 100% healed cortices in coral/MSC vs. autograft, respectively). Given the expense of preparing the coral/MCSs graft and the limitation in cortical bone healing, this is not a viable synthetic substitute for autologous bone graft.

Catros et al25 investigated a synthetic graft composed of hydroxyapatite (HA) and beta-tricalcium phosphate (TCP) in a rabbit subcutaneous model. When IMs were filled with HA-TCP alone, no evidence of new bone formation was found at any time after implantation. However, when autograft was added to the HA-TCP, this allowed for new bone formation and full graft integration by 6 months. Similarly, Bosemark et al51 found that rat femoral defects filled with HA-TCP graft alone showed no healing, whereas 100% healed when BMP-7 was added to the HA-TCP graft. Bone formation further improved with the addition of systemic bisphosphonates at 2 weeks. Therefore, HA-TCP has poor potential as an osteoinductive substance, but it could serve as a graft additive to provide an osteoconductive scaffold in cases where autograft is limited.

Addition of antibiotics to the PMMA spacer has potential benefits about reducing infection rates associated with multiple surgical procedures, as well as helping treat infection when the IM technique is used to fill bone defects exacerbated by posttraumatic osteomyelitis. In a rat femur defect model, Nau et al26 evaluated the effects of using different bone cement spacers with or without the addition of various antibiotics (gentamicin, vancomycin, and clindamycin) on the characteristics of the IM. They concluded that the type of cement and antibiotic additive influenced the membrane thickness and proportion of elastic fibers within the IM, with clindamycin containing spacers having the thinnest membrane at 6 weeks. Shah et al52 studied the effect of clindamycin-impregnated PMMA spacers in rat femoral defects inoculated with Staphylococcus aureus. At 4 weeks, only 1/8 rat treated PMMA spacers supplemented with clindamycin was still infected, whereas 8/8 of rats treated with PMMA spacers without antibiotics remained infected. Furthermore, quantitative polymerase chain reaction analysis of the IMs found that clindamycin did not negatively impact gene expression of inflammatory cytokines, growth factors, and stem cell markers.

Several case series report results of using the IM technique with antibiotic-impregnated PMMA spacers. All 9 posttraumatic bone defects treated in the series by Wong et al5 and all 13 septic nonunions treated in the series by Scholz et al53 went on to uncomplicated union. Twenty-nine of 32 bone defects related to posttraumatic osteomyelitis achieved successful union by 10 months in the series by Wang et al,54 without any cases of recurrent infection. Eleven of 12 cases reported by Apard et al55 went on to union, but not without complications. There were 5 cases of late-onset deep infection in this series, which prompted the authors to conclude that antibiotic spacers do not reduce the rate of infection. Instead, they felt that antibiotic cement might mask incomplete debridements, allowing for infection to develop late in the clinical course.

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AREAS FOR FURTHER RESEARCH

Global Gene Expression of the Membrane

Although specific biological marker levels have been identified as being expressed by the membrane, it is unclear if other factors are involved. A global view of gene expression at various times during the first stage would potentially allow for new, more important factors to become evident. This knowledge could help validate animal models and track changes in the membrane over time. The functional importance of candidate pathways can be tested further in genetically modified animal models or by augmenting or inhibiting the expression of specific genes.

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Alternatives to PMMA

PMMA cement is commonly used in orthopaedics and is readily available. Benefits of using PMMA in the first stage procedure include ability to form and fill the spacer into a defect that can have various shapes and sizes and the ability of PMMA to elute antibiotics into the local environment. However, investigation into alternative substances to fill the segmental defect during the initial operation is lacking. Alternative substances to PMMA (epoxies, silicone, and biosynthetic) could result in improved or expedited production of growth factors and vascularity within the IM. These materials can be tested in animal models before clinical implementation.

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Effects of Antibiotic-Impregnated Cement

Although not originally described as a component of the Masquelet technique, the use of antibiotic-impregnated cement spacers is common. There has been relatively little investigation of the effects of antibiotic-impregnated cement on expression of angiogenic and osteogenic factors. Although selecting appropriate local antibiotic therapy based on bacterial susceptibility would take precedence, having additional information on how these antibiotics affect the physical and molecular properties of the membrane would be extremely valuable and is an area to focus future research.

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Timing

A disadvantage of the Masquelet technique is the staged nature of the surgery and time course needed for full consolidation of new bone. Unfortunately, clinical trials designed to determine an interval between the 2 surgeries that optimizes clinically relevant long-term outcomes (nonunion rates, infection, and reoperations) have not been conducted. Basic science studies suggest that membrane production of osteogenic and angiogenic proteins, cellularity, and vascularity are highest at 4–6 weeks, which supports this as an optimal period for stage 2 grafting. Further basic science research and clinical trials are needed to determine whether this is truly an optimal period.

Alternatively, a lofty goal would be to eliminate the second stage of the procedure all together. With further understanding of the characteristics of the IM, both in terms of structural and biological make-up, it may be possible to produce a synthetic membrane with similar characteristics that can be implanted along with the graft material. This would have to protect the graft from resorption, as well as stimulate bone consolidation. Studies with the long-term goals of optimizing patient outcomes, while decreasing patient morbidity and overall cost of treatment, should be the focus moving forward.

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CONCLUSIONS

The IM technique to address posttraumatic or infected segmental bone loss remains one of the most successful strategies to “fill the defect” in a way that is length independent, technically reproducible, patient friendly, and cost effective compared with other modalities as long as a thorough debridement has been performed. The multiple variables highlighted in this article and their implications on the quality of the membrane and success of treatment qualify the Masquelet technique as an art more than a science. It is critical to focus our research on the many questions raised in this manuscript and compile standards and treatment algorithms that may help produce quality prospective research on the topic.

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Keywords:

induced membrane; Masquelet technique; basic science of induced membrane; growth factors; segmental bone loss; critical defect bone reconstruction

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