Inhibitory SMAD6 interferes with BMP-dependent generation of muscle progenitor cells and perturbs proximodistal pattern of murine limb muscles.

The mechanism of pattern formation during limb muscle development remains poorly understood. The canonical view holds that naïve limb muscle progenitor cells (MPCs) invade a pre-established pattern of muscle connective tissue, thereby forming individual muscles. Here, we show that early murine embryonic limb MPCs highly accumulate pSMAD1/5/9, demonstrating active signaling of bone morphogenetic proteins (BMP) in these cells. Overexpression of inhibitory human SMAD6 (huSMAD6) in limb MPCs abrogated BMP signaling, impaired their migration and proliferation, and accelerated myogenic lineage progression. Fewer primary myofibers developed, causing an aberrant proximodistal muscle pattern. Patterning was not disturbed when huSMAD6 was overexpressed in differentiated muscle, implying that the proximodistal muscle pattern depends on BMP-mediated expansion of MPCs before their differentiation. We show that limb MPCs differentially express Hox genes, and Hox-expressing MPCs displayed active BMP signaling. huSMAD6 overexpression caused loss of HOXA11 in early limb MPCs. In conclusion, our data show that BMP signaling controls expansion of embryonic limb MPCs as a prerequisite for establishing the proximodistal muscle pattern, a process that involves expression of Hox genes.

TMEM8C-mediated fusion is regionalized and regulated by NOTCH signalling during foetal myogenesis.

The location and regulation of fusion events within skeletal muscles during development remain unknown. Using the fusion marker myomaker (Mymk), named TMEM8C in chicken, as a readout of fusion, we identified a co-segregation of TMEM8C-positive cells and MYOG-positive cells in single-cell RNA-sequencing datasets of limbs from chicken embryos. We found that TMEM8C transcripts, MYOG transcripts and the fusion-competent MYOG-positive cells were preferentially regionalized in central regions of foetal muscles. We also identified a similar regionalization for the gene encoding the NOTCH ligand JAG2 along with an absence of NOTCH activity in TMEM8C+ fusion-competent myocytes. NOTCH function in myoblast fusion had not been addressed so far. We analysed the consequences of NOTCH inhibition for TMEM8C expression and myoblast fusion during foetal myogenesis in chicken embryos. NOTCH inhibition increased myoblast fusion and TMEM8C expression and released the transcriptional repressor HEYL from the TMEM8C regulatory regions. These results identify a regionalization of TMEM8C-dependent fusion and a molecular mechanism underlying the fusion-inhibiting effect of NOTCH in foetal myogenesis. The modulation of NOTCH activity in the fusion zone could regulate the flux of fusion events.

Genome-wide strategies identify downstream target genes of chick connective tissue-associated transcription factors.

Connective tissues support organs and play crucial roles in development, homeostasis and fibrosis, yet our understanding of their formation is still limited. To gain insight into the molecular mechanisms of connective tissue specification, we selected five zinc-finger transcription factors - OSR1, OSR2, EGR1, KLF2 and KLF4 - based on their expression patterns and/or known involvement in connective tissue subtype differentiation. RNA-seq and ChIP-seq profiling of chick limb micromass cultures revealed a set of common genes regulated by all five transcription factors, which we describe as a connective tissue core expression set. This common core was enriched with genes associated with axon guidance and myofibroblast signature, including fibrosis-related genes. In addition, each transcription factor regulated a specific set of signalling molecules and extracellular matrix components. This suggests a concept whereby local molecular niches can be created by the expression of specific transcription factors impinging on the specification of local microenvironments. The regulatory network established here identifies common and distinct molecular signatures of limb connective tissue subtypes, provides novel insight into the signalling pathways governing connective tissue specification, and serves as a resource for connective tissue development.

EGR1 Transcription Factor is a Multifaceted Regulator of Matrix Production in Tendons and Other Connective Tissues.

Although the transcription factor EGR1 is known as NGF1-A, TIS8, Krox24, zif/268, and ZENK, it still has many fewer names than biological functions. A broad range of signals induce Egr1 gene expression via numerous regulatory elements identified in the Egr1 promoter. EGR1 is also the target of multiple post-translational modifications, which modulate EGR1 transcriptional activity. Despite the myriad regulators of Egr1 transcription and translation, and the numerous biological functions identified for EGR1, the literature reveals a recurring theme of EGR1 transcriptional activity in connective tissues, regulating genes related to the extracellular matrix. Egr1 is expressed in different connective tissues, such as tendon (a dense connective tissue), cartilage and bone (supportive connective tissues), and adipose tissue (a loose connective tissue). Egr1 is involved in the development, homeostasis, and healing processes of these tissues, mainly via the regulation of extracellular matrix. In addition, Egr1 is often involved in the abnormal production of extracellular matrix in fibrotic conditions, and Egr1 deletion is seen as a target for therapeutic strategies to fight fibrotic conditions. This generic EGR1 function in matrix regulation has little-explored implications but is potentially important for tendon repair.

TGFß and FGF promote tendon progenitor fate and act downstream of muscle contraction to regulate tendon differentiation during chick limb development.

The molecular programme underlying tendon development has not been fully identified. Interactions with components of the musculoskeletal system are important for limb tendon formation. Limb tendons initiate their development independently of muscles; however, muscles are required for further tendon differentiation. We show that both FGF/ERK MAPK and TGFß/SMAD2/3 signalling pathways are required and sufficient for SCX expression in chick undifferentiated limb cells, whereas the FGF/ERK MAPK pathway inhibits Scx expression in mouse undifferentiated limb mesodermal cells. During differentiation, muscle contraction is required to maintain SCX, TNMD and THBS2 expression in chick limbs. The activities of FGF/ERK MAPK and TGFß/SMAD2/3 signalling pathways are decreased in tendons under immobilisation conditions. Application of FGF4 or TGFß2 ligands prevents SCX downregulation in immobilised limbs. TGFß2 but not FGF4 prevent TNMD and THBS2 downregulation under immobilisation conditions. We did not identify any intracellular crosstalk between both signalling pathways in their positive effect on SCX expression. Independently of each other, both FGF and TGFß promote tendon commitment of limb mesodermal cells and act downstream of mechanical forces to regulate tendon differentiation during chick limb development.

Transcriptomic analysis of mouse limb tendon cells during development.

The molecular signals driving tendon development are not fully identified. We have undertaken a transcriptome analysis of mouse limb tendon cells that were isolated at different stages of development based on scleraxis (Scx) expression. Microarray comparisons allowed us to establish a list of genes regulated in tendon cells during mouse limb development. Bioinformatics analysis of the tendon transcriptome showed that the two most strongly modified signalling pathways were TGF-ß and MAPK. TGF-ß/SMAD2/3 gain- and loss-of-function experiments in mouse limb explants and mesenchymal stem cells showed that TGF-ß signalling was sufficient and required via SMAD2/3 to drive mouse mesodermal stem cells towards the tendon lineage ex vivo and in vitro. TGF-ß was also sufficient for tendon gene expression in late limb explants during tendon differentiation. FGF does not have a tenogenic effect and the inhibition of the ERK MAPK signalling pathway was sufficient to activate Scx in mouse limb mesodermal progenitors and mesenchymal stem cells.

Specific pattern of cell cycle during limb fetal myogenesis.

Tight regulation of cell proliferation and differentiation is required to ensure proper growth during development and post-natal life. The source and nature of signals regulating cell proliferation are not well identified in vivo. We investigated the specific pattern of proliferating cells in mouse limbs, using the Fluorescent ubiquitynation-based cell-cycle indicator (Fucci) system, which allowed the visualization of the G1, G1/S transition and S/G2/M phases of the cell cycle in red, yellow or green fluorescent colors, respectively. We also used the retroviral RCAS system to express a Fucci cassette in chick embryos. We performed a comprehensive analysis of the cell cycle state of myogenic cells in fetal limb muscles, adult myoblast primary cultures and isolated muscle fiber cultures using the Fucci transgenic mice. We found that myonuclei of terminally differentiated muscle fibers displayed Fucci red fluorescence during mouse and chick fetal development, in adult isolated muscle fiber (ex vivo) and adult myoblast (in vitro) mouse cultures. This indicated that myonuclei exited from the cell cycle in the G1 phase and are maintained in a blocked G1-like state. We also found that cycling muscle progenitors and myoblasts in G1 phase were not completely covered by the Fucci system. During mouse fetal myogenesis, Pax7+ cells labeled with the Fucci system were observed mostly in S/G2/M phases. Proliferating cells in S/G2/M phases displayed a specific pattern in mouse fetal limbs, delineating individualized muscles. In addition, we observed more Pax7+ cells in S/G2/M phases at muscle tips, compared to the middle of muscles. These results highlight a specific spatial regionalization of cycling cells at the muscle borders and muscle-tendon interface during fetal development.

Stable and bicistronic expression of two genes in somite- and lateral plate-derived tissues to study chick limb development.

BACKGROUND: Components of the limb musculoskeletal system have distinct mesoderm origins. Limb skeletal muscles originate from somites, while the skeleton and attachments (tendons and connective tissues) derive from limb lateral plate. Despite distinct mesoderm origins, the development of muscle, skeleton and attachments is highly coordinated both spatially and temporally to ensure complete function of the musculoskeletal system. A system to study molecular interactions between somitic-derived tissues (muscles) and lateral-plate-derived tissues (skeletal components and attachments) during limb development is missing. RESULTS: We designed a gene delivery system in chick embryos with the ultimate aim to study the interactions between the components of the musculoskeletal system during limb development. We combined the Tol2 genomic integration system with the viral T2A system and developed new vectors that lead to stable and bicistronic expression of two proteins at comparable levels in chick cells. Combined with limb somite and lateral plate electroporation techniques, two fluorescent reporter proteins were co-expressed in stoichiometric proportion in the muscle lineage (somitic-derived) or in skeleton and their attachments (lateral-plate-derived). In addition, we designed three vectors with different promoters to target muscle cells at different steps of the differentiation process. CONCLUSION: Limb somite electroporation technique using vectors containing these different promoters allowed us to target all myogenic cells, myoblasts or differentiated muscle cells. These stable and promoter-specific vectors lead to bicistronic expression either in somitic-derived myogenic cells or lateral plate-derived cells, depending on the electroporation sites and open new avenues to study the interactions between myogenic cells and tendon or connective tissue cells during limb development.

Sim2 prevents entry into the myogenic program by repressing MyoD transcription during limb embryonic myogenesis.

The basic helix-loop-helix transcription factor MyoD is a central actor that triggers the skeletal myogenic program. Cell-autonomous and non-cell-autonomous regulatory pathways must tightly control MyoD expression to ensure correct initiation of the muscle program at different places in the embryo and at different developmental times. In the present study, we have addressed the involvement of Sim2 (single-minded 2) in limb embryonic myogenesis. Sim2 is a bHLH-PAS transcription factor that inhibits transcription by active repression and displays enhanced expression in ventral limb muscle masses during chick and mouse embryonic myogenesis. We have demonstrated that Sim2 is expressed in muscle progenitors that have not entered the myogenic program, in different experimental conditions. MyoD expression is transiently upregulated in limb muscle masses of Sim2(-/-) mice. Conversely, Sim2 gain-of-function experiments in chick and Xenopus embryos showed that Sim2 represses MyoD expression. In addition, we show that Sim2 represses the activity of the mouse MyoD promoter in primary myoblasts and is recruited to the MyoD core enhancer in embryonic mouse limbs. Sim2 expression is non-autonomously and negatively regulated by the dorsalising factor Lmx1b. We propose that Sim2 represses MyoD transcription in limb muscle masses, through Sim2 recruitment to the MyoD core enhancer, in order to prevent premature entry into the myogenic program. This MyoD repression is predominant in ventral limb regions and is likely to contribute to the differential increase of the global mass of ventral muscles versus dorsal muscles.

Bioinspired scaffolds based on aligned polyurethane nanofibers mimic tendon and ligament fascicles.

Topographical factors of scaffolds play an important role in regulating cell functions. Although the effects of alignment topography and three-dimensional (3D) configuration of nanofibers as well as surface stiffness on cell behavior have been investigated, there are relatively few reports that attempt to understand the relationship between synergistic effects of these parameters and cell responses. Herein, the influence of biophysical and biomechanical cues of electrospun polyurethane (PU) scaffolds on mesenchymal stem cells (MSCs) activities was evaluated. To this aim, multiscale bundles were developed by rolling up the aligned electrospun mats mimicking the fascicles of tendons/ligaments and other similar tissues. Compared to mats, the 3D bundles not only maintained the desirable topographical features (i.e., fiber diameter, fiber orientation, and pore size), but also boosted tensile strength (∼40 MPa), tensile strain (∼260%), and surface stiffness (∼1.75 MPa). Alignment topography of nanofibers noticeably dictated cell elongation and a uniaxial orientation, resulting in tenogenic commitment of MSCs. MSCs seeded on the bundles expressed higher levels of tenogenic markers compared to mats. Moreover, the biomimetic bundle scaffolds improved synthesis of extracellular matrix components compared to mats. These results suggest that biophysical and biomechanical cues modulate cell-scaffold interactions, providing new insights into hierarchical scaffold design for further studies.

EGR1 and EGR2 involvement in vertebrate tendon differentiation.

The molecules involved in vertebrate tendon formation during development remain largely unknown. To date, only two DNA-binding proteins have been identified as being involved in vertebrate tendon formation, the basic helix-loop-helix transcription factor Scleraxis and, recently, the Mohawk homeobox gene. We investigated the involvement of the early growth response transcription factors Egr1 and Egr2 in vertebrate tendon formation. We established that Egr1 and Egr2 expression in tendon cells was correlated with the increase of collagen expression during tendon cell differentiation in embryonic limbs. Vertebrate tendon differentiation relies on a muscle-derived FGF (fibroblast growth factor) signal. FGF4 was able to activate the expression of Egr genes and that of the tendon-associated collagens in chick limbs. Egr gene misexpression experiments using the chick model allowed us to establish that either Egr gene has the ability to induce de novo expression of the reference tendon marker scleraxis, the main tendon collagen Col1a1, and other tendon-associated collagens Col3a1, Col5a1, Col12a1, and Col14a1. Mouse mutants for Egr1 or Egr2 displayed reduced amounts of Col1a1 transcripts and a decrease in the number of collagen fibrils in embryonic tendons. Moreover, EGR1 and EGR2 trans-activated the mouse Col1a1 proximal promoter and were recruited to the tendon regulatory regions of this promoter. These results identify EGRs as novel DNA-binding proteins involved in vertebrate tendon differentiation by regulating type I collagen production.

Stable, conditional, and muscle-fiber-specific expression of electroporated transgenes in chick limb muscle cells.

Limb muscle formation is spread out over time and, consequently, muscle cells are not easy to target at any particular stages. We aimed to design a technique to study gene function in the different steps of limb muscle formation. We have associated transposon-mediated gene transfer and a tetracycline-dependent activation method with forelimb somite electroporation. In addition, we have established a new vector combining a differentiated-muscle-cell-specific promoter and the transposon system, which allows stable gene expression in limb differentiated muscle cells and not in muscle progenitors. Using these methods, we observed that conditional Fgf4 expression in muscle cells at the onset of fetal muscle differentiation and specific Fgf4 expression in differentiated muscle cells alter muscle fiber formation in chick limbs. Limb somite electroporation with these set of vectors allowing stable, conditional, and differentiated-muscle-specific expression of transgenes opens new perspectives for investigating gene function at various steps of limb muscle formation.

Core-Shell Pure Collagen Threads Extruded from Highly Concentrated Solutions Promote Colonization and Differentiation of C3H10T1/2 Cells.

The elaboration of scaffolds able to efficiently promote cell differentiation toward a given cell type remains challenging. Here, we engineered dense type I collagen threads with the aim of providing scaffolds with specific morphological and mechanical properties for C3H10T1/2 mesenchymal stem cells. Extrusion of pure collagen solutions at different concentrations (15, 30, and 60 mg/mL) in a PBS 5× buffer generated dense fibrillated collagen threads. For the two highest concentrations, threads displayed a core-shell structure with a marked fibril orientation of the outer layer along the longitudinal axis of the threads. Young's modulus and ultimate tensile stress as high as 1 and 0.3 MPa, respectively, were obtained for the most concentrated collagen threads without addition of any cross-linkers. C3H10T1/2 cells oriented themselves with a mean angle of 15-24° with respect to the longitudinal axis of the threads. Cells penetrated the 30 mg/mL scaffolds but remained on the surface of the 60 mg/mL ones. After three weeks of culture, cells displayed strong expression of the tendon differentiation marker Tnmd, especially for the 30 mg/mL threads. These results suggest that both the morphological and mechanical characteristics of collagen threads are key factors in promoting C3H10T1/2 differentiation into tenocytes, offering promising levers to optimize tissue engineering scaffolds for tendon regeneration.

Unexpected contribution of fibroblasts to muscle lineage as a mechanism for limb muscle patterning.

Positional information driving limb muscle patterning is contained in connective tissue fibroblasts but not in myogenic cells. Limb muscles originate from somites, while connective tissues originate from lateral plate mesoderm. With cell and genetic lineage tracing we challenge this model and identify an unexpected contribution of lateral plate-derived fibroblasts to the myogenic lineage, preferentially at the myotendinous junction. Analysis of single-cell RNA-sequencing data from whole limbs at successive developmental stages identifies a population displaying a dual muscle and connective tissue signature. BMP signalling is active in this dual population and at the tendon/muscle interface. In vivo and in vitro gain- and loss-of-function experiments show that BMP signalling regulates a fibroblast-to-myoblast conversion. These results suggest a scenario in which BMP signalling converts a subset of lateral plate mesoderm-derived cells to a myogenic fate in order to create a boundary of fibroblast-derived myonuclei at the myotendinous junction that controls limb muscle patterning.

Egr1 loss-of-function promotes beige adipocyte differentiation and activation specifically in inguinal subcutaneous white adipose tissue.

In mice, exercise, cold exposure and fasting lead to the differentiation of inducible-brown adipocytes, called beige adipocytes, within white adipose tissue and have beneficial effects on fat burning and metabolism, through heat production. This browning process is associated with an increased expression of the key thermogenic mitochondrial uncoupling protein 1, Ucp1. Egr1 transcription factor has been described as a regulator of white and beige differentiation programs, and Egr1 depletion is associated with a spontaneous increase of subcutaneous white adipose tissue browning, in absence of external stimulation. Here, we demonstrate that Egr1 mutant mice exhibit a restrained Ucp1 expression specifically increased in subcutaneous fat, resulting in a metabolic shift to a more brown-like, oxidative metabolism, which was not observed in other fat depots. In addition, Egr1 is necessary and sufficient to promote white and alter beige adipocyte differentiation of mouse stem cells. These results suggest that modulation of Egr1 expression could represent a promising therapeutic strategy to increase energy expenditure and to restrain obesity-associated metabolic disorders.

Long-term hydrolytic degradation study of polycaprolactone films and fibers grafted with poly(sodium styrene sulfonate): Mechanism study and cell response.

Polycaprolactone (PCL) is a widely used biodegradable polyester for tissue engineering applications when long-term degradation is preferred. In this article, we focused on the analysis of the hydrolytic degradation of virgin and bioactive poly(sodium styrene sulfonate) (pNaSS) functionalized PCL surfaces under simulated physiological conditions (phosphate buffer saline at 25 and 37 °C) for up to 120 weeks with the aim of applying bioactive PCL for ligament tissue engineering. Techniques used to characterize the bulk and surface degradation indicated that PCL was hydrolyzed by a bulk degradation mode with an accelerated degradation-three times increased rate constant-for pNaSS grafted PCL at 37 °C when compared to virgin PCL at 25 °C. The observed degradation mechanism is due to the pNaSS grafting process (oxidation and radical polymerization), which accelerated the degradation until 48 weeks, when a steady state is reached. The PCL surface was altered by pNaSS grafting, introducing hydrophilic sulfonate groups that increase the swelling and smoothing of the surface, which facilitated the degradation. After 48 weeks, pNaSS was largely removed from the surface, and the degradation of virgin and pNaSS grafted surfaces was similar. The cell response of primary fibroblast cells from sheep ligament was consistent with the surface analysis results: a better initial spreading of cells on pNaSS surfaces when compared to virgin surfaces and a tendency to become similar with degradation time. It is worthy to note that during the extended degradation process the surfaces were able to continue inducing better cell spreading and preserve their cell phenotype as shown by collagen gene expressions.

Mechanical and molecular parameters that influence the tendon differentiation potential of C3H10T1/2 cells in 2D- and 3D-culture systems.

One of the main challenges relating to tendons is to understand the regulators of the tendon differentiation program. The optimum culture conditions that favor tendon cell differentiation have not been identified. Mesenchymal stem cells present the ability to differentiate into multiple lineages in cultures under different cues ranging from chemical treatment to physical constraints. We analyzed the tendon differentiation potential of C3H10T1/2 cells, a murine cell line of mesenchymal stem cells, upon different 2D- and 3D-culture conditions. We observed that C3H10T1/2 cells cultured in 2D conditions on silicone substrate were more prone to tendon differentiation, assessed with the expression of the tendon markers Scx, Col1a1 and Tnmd as compared to cells cultured on plastic substrate. The 3D-fibrin environment was more favorable for Scx and Col1a1 expression compared to 2D cultures. We also identified TGFß2 as a negative regulator of Tnmd expression in C3H10T1/2 cells in 2D and 3D cultures. Altogether, our results provide us with a better understanding of the culture conditions that promote tendon gene expression and identify mechanical and molecular parameters upon which we could act to define the optimum culture conditions that favor tenogenic differentiation in mesenchymal stem cells.

A molecular clock operates during chick autopod proximal-distal outgrowth.

Temporal control can be considered the fourth dimension in embryonic development. The identification of the somitogenesis molecular clock provided new insight into how embryonic cells measure time. We provide the first evidence of a molecular clock operating during chick fore-limb autopod outgrowth and patterning, by showing that the expression of the somitogenesis clock component hairy2 cycles in autopod chondrogenic precursor cells with a 6 h periodicity. We determined the length of time required to form an autopod skeletal limb element, and established a correlation between the latter and the periodicity of cyclic hairy2 gene expression. We suggest that temporal control exerted by cyclic gene expression can be a widespread mechanism providing cellular temporal information during vertebrate embryonic development.

A dual transcript-discovery approach to improve the delimitation of gene features from RNA-seq data in the chicken model.

The sequence of the chicken genome, like several other draft genome sequences, is presently not fully covered. Gaps, contigs assigned with low confidence and uncharacterized chromosomes result in gene fragmentation and imprecise gene annotation. Transcript abundance estimation from RNA sequencing (RNA-seq) data relies on read quality, library complexity and expression normalization. In addition, the quality of the genome sequence used to map sequencing reads, and the gene annotation that defines gene features, must also be taken into account. A partially covered genome sequence causes the loss of sequencing reads from the mapping step, while an inaccurate definition of gene features induces imprecise read counts from the assignment step. Both steps can significantly bias interpretation of RNA-seq data. Here, we describe a dual transcript-discovery approach combining a genome-guided gene prediction and a de novo transcriptome assembly. This dual approach enabled us to increase the assignment rate of RNA-seq data by nearly 20% as compared to when using only the chicken reference annotation, contributing therefore to a more accurate estimation of transcript abundance. More generally, this strategy could be applied to any organism with partial genome sequence and/or lacking a manually-curated reference annotation in order to improve the accuracy of gene expression studies.

Poly(ε-caprolactone)/Hydroxyapatite 3D Honeycomb Scaffolds for a Cellular Microenvironment Adapted to Maxillofacial Bone Reconstruction.

The elaboration of biomimetic materials inspired from the specific structure of native bone is one the main goal of tissue engineering approaches. To offer the most appropriate environment for bone reconstruction, we combined electrospinning and electrospraying to elaborate an innovative scaffold composed of alternating layers of polycaprolactone (PCL) and hydroxyapatite (HA). In our approach, the electrospun PCL was shaped into a honeycomb-like structure with an inner diameter of 160 µm, capable of providing bone cells with a 3D environment while ensuring the material biomechanical strength. After 5 days of culture without any differentiation factor, the murine embryonic cell line demonstrated excellent cell viability on contact with the PCL-HA structures as well as active colonization of the scaffold. The cell differentiation, as tested by RT-qPCR, revealed a 6-fold increase in the expression of the RNA of the Bglap involved in bone mineralization as compared to a classical 2D culture. This differentiation of the cells into osteoblasts was confirmed by alkaline phosphatase staining of the scaffold cultivated with the cell lineage. Later on, organotypic cultures of embryonic bone tissues showed the high capacity of the PCL-HA honeycomb structure to guide the migration of differentiated bone cells throughout the cavities and the ridge of the biomaterial, with a colonization surface twice as big as that of the control. Taken together, our results indicate that PCL-HA honeycomb structures are biomimetic supports that promotes in vitro osteocompatibility, osteoconduction, and osteoinduction and could be suitable for being used for bone reconstruction in complex situations such as the repair of maxillofacial defects.