Use of the Induced Membrane Technique for Bone Tissue Engineering Purposes: Animal Studies

Animal experiments using the induced membrane procedure for bone tissue engineering purposes have provided evidence that the membrane has structural characteristics and biologic properties that may be used for bone tissue engineering purposes. Clinically relevant animal models have demonstrated that standardized particulate bone constructs can be used to repair large bone defects using the procedure and that the osteogenic ability of these constructs partially approaches that of bone autografts.

Clinical management of extensive segmental long-bone defects occurring after high-energy trauma or debridement for infected nonunion poses a substantial orthopedic challenge. Current solutions include the use of cancellous autografts when defects are 4 to 5 cm or smaller, whereas other procedures such as transfer of vascularized bone or intercalary bone transport methods are required to treat longer defects.

Masquelet and colleagues reported successful repair of wide diaphyseal defects (≤25 cm) with concurrent severe soft tissue loss in human patients using fresh autologous cancellous bone grafts in an original two-step surgical procedure. In the first step, a polymethylmethacrylate (PMMA) cement spacer was inserted into the defect inducing formation of an encapsulation membrane. After 8 weeks, the spacer was removed and the cavity filled with autologous cancellous graft harvested from the iliac crest. Bone union was usually achieved within 8.5 months, with patients recovering normal gait and motion. Interestingly, the procedure applied to all sorts of defects in contrast to the use of a massive allograft. Clinical trials in which the procedure has been performed have highlighted the advantages of using the induced membrane: (1) prevention of protrusion of adjacent soft tissues in the bone defect; (2) restraint of the graft in place; and, according to Masquelet, (3) maintenance of graft volume over time through either protection of the graft against resorption or local production of osteoinductive substances. Interestingly, animal studies have demonstrated that the membrane indeed has histologic characteristics and biologic properties that might facilitate bone healing.

Large quantities of autograft and sometimes repeated surgery to achieve bone union are nevertheless needed for successful outcome with this procedure. The development of tissue-engineered bone constructs (TEBC) combining osteoconductive scaffolds with autologous mesenchymal stem cells (MSC), the availability of bone morphogenetic proteins, have opened new avenues for designing efficient bone substitutes. The possibility of placing either skeletal stem cells or bone morphogenetic proteins (BMPs) onto scaffolds in the cavity delineated by the membrane is a very attractive prospect.

The purpose of this article was to review data from animal studies that have allowed better understanding of the structure and properties of the membrane as well as the preliminary and preclinical evaluations of new therapeutic strategies using the induced membrane procedure.

Animal models using the induced membrane procedure

The structure and biologic properties of the induced membrane have been studied in small (rabbit) and large (sheep) animal species in which implantation of PMMA cylinders in either the dorsal paraspinal subcutaneous tissue (ectopic implantation sites) or segmental bone defects (orthotopic implantation sites) have induced the formation of a membrane. These animal models have been further used to assess the osteogenic potential of different TEBC.

Ectopic Models

Subcutaneous implantation of PMMA cylinders (four 10-mm-diameter, 5-mm-long cylinders in rabbits, eighteen 15-mm-diameter, 28-mm-long cylinders in sheep), have made possible the analysis of the structure and biologic characteristics of the induced membrane at different time points after cement implantation.

Briefly, induced membranes were sampled at different time points postoperatively (2, 4, 6, and 8 weeks in rabbit experiments; 6 and 14 weeks in sheep experiments) and processed for histology and immunohistochemical analysis to assess structure and inflammatory reaction. Samples from membranes induced in sheep were immunostained to assess the presence of Col-1 and vascular endothelial growth factor (VEGF), as well as CD14 (for macrophages) and CBFA-1 (for osteoblast precursor cells) positive cells. Samples from membranes induced in rabbits were processed for protein extraction and dosage of growth factors (VEGF, transforming growth factor [TGF]-β1, BMP-2). Proliferation and differentiation of human bone marrow stromal cells grown into a culture medium containing protein extracts from either the induced membrane or subcutaneous tissue (controls) were also evaluated.

Orthotopic Models

Implantation of PMMA cylinders in either 25-mm-long metatarsal or 30-mm-long femoral segmental bone critical size bone defects (CSD) stabilized with a bone plate in sheep has made possible the validation of the procedure described by Masquelet and colleagues using morselized corticocancellous bone autograft in conditions similar to the one encountered in clinical situations (eg, replacement of a CSD in a load-bearing bone, in an animal with bone healing and remodeling characteristics close to humans’). Briefly, a mid-diaphyseal segmental bone resection was created and filled with a cement spacer for 4 or 6 weeks. At these time points, the cement was carefully removed through an incision made along the formed encapsulation membrane surrounding the cement, and the resultant cavities were either left empty or filled with a morselized autologous bone graft sampled from the iliac crest. Bone healing was obtained in both femoral and metatarsal defects within 4 to 6 months, respectively. Membrane induced at the metatarsal site was sampled and processed for histology and immunohistochemistry at time of cement removal and bone grafting, 6 weeks postoperatively, and at the time the animals were humanely killed 6 months later, making possible the characterization of its structure in a bone site.

Characteristics and biologic properties of the induced membrane: data from animal experiments

Macroscopic Characteristics

Both subcutaneous and orthotopic implantation of PMMA cylinders in rabbit and sheep experiments induced the formation of a fibrous membrane. Implantation in ectopic and orthotopic sites in sheep have shown that at time of cement removal, 6 weeks after cement implantation, the membrane was 1 to 2 mm thick, not adherent to underlying cement thus allowing easy removal of the later and bled when incised. It was also mechanically competent in both locations and incised edges could be sutured without tension thus delineating a cavity in which either bone autograft or TEBC could be contained.

As shown in sheep in which PMMA cylinders were implanted into segmental metatarsal bone defects, the encapsulation membrane prevented adjacent soft tissue protrusion in the defect. In these experiments, the membrane was indeed found adherent to the resected bone edges and did not collapse after cement removal, thus delineating a cavity corresponding to the volume of the retrieved cement spacer. This was an important outcome because it has been reported that the use of pliable and nonrigid membranes (ie, silicone sheeting) across a defect may result in nonunions because of collapse of the membrane and subsequent interposition of tissue in the defect.

Interestingly, the membrane defined the shape of the regenerating bone as replacement bone retained the original size and contour along the defect throughout the study. The induced membrane indeed constrained the autograft, which was a critical issue, as in human patients, a major problem encountered with use of morselized graft for segmental bone losses greater than 4 cm long is constraint of bone chips to the site and prevention of soft tissue protrusion into the defect.

At last, use of an enclosed space to constrain autologous bone chips within the defect prevented ectopic bone formation. This is of particular relevance in tissue engineering strategies where maintenance of biologic agents within defect areas is critical.

Histologic and Immunohistochemical Characteristics

Histologic and immunohistochemical analysis of membrane specimens sampled 6 weeks after subcutaneous or orthotopical implantation of PMMA cylinders in both rabbit and sheep models have shown that the induced membrane was made of a collagenous matrix in which numerous elongated fibroblasticlike cells were found embedded ( Fig. 1 ). Collagen fibers were orientated parallel to the PMMA surface.