Periosteum-guided prefabrication of vascularized bone of clinical shape and volume.

BACKGROUND: Large craniofacial skeletal defects require complex reconstruction. Vascularized tissue transfer is the current standard in treatment, but these operations are technically difficult and associated with donor-site morbidity. Guided flap prefabrication offers a technique for endogenously engineering vascularized composite tissues with complex three-dimensional structure. This study evaluates the relationship between implantation time and tissue structure for generating tissues of clinically relevant volume and structure. METHODS: Twenty skeletally mature domestic sheep were implanted with poly(methyl methacrylate) chambers designed to mimic the size and shape of the mental protuberance of the mandible. Each chamber was filled with morcellized bone graft and implanted with the open face apposed to the cambium layer of the rib periosteum. Chambers were harvested at 3, 6, 9, 12, and 24 weeks, and the tissue inside the chambers was analyzed for shape conformation to chamber geometry, gross tissue volume, and bone histomorphometric parameters. RESULTS: Histologically, active endochondral, direct, and appositional bone formation was observed. Calcified tissue area and new bone formation increased for each time point up to 12 weeks of implantation. The tissues formed maintained volumetric and geometrical structure consistent with the chamber up to 9 weeks after implantation. Significant decreases in total volume and agreement with chamber geometry were observed at 12 and 24 weeks. CONCLUSIONS: Periosteum-guided tissue prefabrication was found to be an effective means of engineering three-dimensional vascularized bone of clinical size and shape. The optimal duration of incubation before significant volume loss occurs is 9 weeks in this large-animal model.

Use of vascularized periosteum or bone to improve healing of segmental allografts after tumor resection: an ovine rib model.

BACKGROUND: Despite technical advances, nonunion or delayed union remains an important clinical problem when segmental allografts are used to repair diaphyseal defects after bone tumor resection. Using an ovine rib model, the authors studied whether the addition of a vascularized periosteum or bone flap improved healing compared with a segmental allograft alone. METHODS: A 4-cm segment of rib was resected from four consecutive ribs of 15 sheep. Three different reconstructions were compared within the same sheep: allograft alone, allograft and vascularized periosteum, and allograft and vascularized bone. One defect was not reconstructed and served as a control. Five sheep were humanely killed at each of the following time points: 9, 12, and 15 weeks. The host-allograft junctions were analyzed using plain radiographs, micro-computed tomography, and histologic examination. RESULTS: Micro-computed tomographic analysis showed significant improvement with each reconstruction technique over time. Plain radiographs and histologic analyses demonstrated earlier bridging of the host-allograft junction when either vascularized periosteum or vascularized bone was used compared with allograft alone. CONCLUSION: Use of vascularized periosteum or bone may facilitate healing of the host-allograft junction after intercalary allograft reconstruction.

Animal models for adipose tissue engineering.

There is a critical need for adequate reconstruction of soft tissue defects resulting from tumor resection, trauma, and congenital abnormalities. To be sure, adipose tissue engineering strategies offer promising solutions. However, before clinical translation can occur, efficacy must be proven in animal studies. The aim of this review is to provide an overview of animal models currently employed for adipose tissue engineering.

Micro-well texture printed into PEG hydrogels using the FILM nanomanufacturing process affects the behavior of preadipocytes.

To date, biomaterial scaffolds for adipose tissue engineering have focused on macro- and upper micro-scale fabrication, biocompatibility, and biodegradation, but have failed to recapitulate the sub-micron dimensions of native extracellular matrix (ECM) and, therefore, have not optimized material-cell interactions. Here, we report the findings from a study investigating the effects of a quasi-mimetic sub-micron (< 1 micrometer) surface texture on the qualitative behavior of preadipocytes (PAs). We found that PAs in contact with tread-like micro-well structures exhibit a different morphology relative to PAs seeded onto control smooth glass surfaces. Additionally, the micro-well topography induced isolated PAs to undergo adipogenesis, which usually occurs in the presence of aggregates of contact-inhibited PAs. The micro-well structures were printed into polyethylene glycol dimethacrylate (PEGDMA) using the recently reported nanomanufacturing process called Flash Imprint Lithography Using a Mask Aligner (FILM). FILM is a simple process that can be utilized to fabricate micro- and nanostructures in UV-curable materials (D.Y. Fozdar, W. Zhang, M. Palard, C.W. Patrick Jr., S.C. Chen, Flash Imprint Lithography Using A Mask Aligner (FILM): A Method for Printing Nanostructures in Photosensitive Hydrogels for Tissue Engineering. Nanotechnology 19, 2008). We demonstrate the utilization of the FILM process for a tissue engineering application for the first time. The micro-well topographical theme is characterized by contact angle and surface energy analysis and the results were compared with those for smooth glass and unpatterned PEGDMA surfaces. Based on our observations, we believe that the micro-well texture may ultimately be beneficial on implantable tissue scaffolds.

Quantitative analysis of the microvasculature growing in the fibrin interface between a skin graft and the recipient site.

Current tissue engineering techniques have failed to provide an established microvasculature in skin substitutes, a requisite for the maintenance of graft viability and rapid revascularization subsequent to graft transplantation in vivo. To improve outcomes for both conventional skin grafts and skin substitutes, the existing knowledge gap concerning the spatio-temporal mechanisms of skin graft revascularization must be abrogated. The current knowledge gap is due, at least in part, to a lack of appropriate diagnostic methods to quantify skin graft revascularization. To enhance the understanding of skin graft revascularization, we quantitatively evaluated revascularization of autologous skin grafts in a rat model by quantifying 2- and 3-dimensional vascular metrics in the fibrin interface 3, 7, and 10 days after transplantation. In this study, the fibrin interface appeared to be completely replaced with fibrovascular tissue by postoperative day 10. Although the mean vessel diameter was about 10 mum for the time points sampled, the mean vessel number, area, and volume fraction increased about 2.5-fold from postoperative day 3 to 7 and then decreased about 1.27-fold at postoperative day 10. There was no significant difference between 2- and 3-dimensional vascular metrics based on Bland-Altman analysis. In conclusion, these data establish a standard for metrics of vessels growing in the fibrin interface of a rat autologous skin graft and its donor site and suggests that once the blood supply has been restored to a viable transplant, the number, area, and volume fractions of vessels decrease to levels found at postoperative day 3.

Comparison of guided bone formation from periosteum and muscle fascia.

BACKGROUND: Muscle fascia and periosteum have been used clinically to guide prefabrication of vascularized bone flaps for reconstruction of complex three-dimensional tissues. Although it seems that both locations have the capacity to generate vascularized bone, there have been no studies that directly compare different implantation sites. The authors performed a rigorous, quantitative, histomorphometric comparison of bone prefabrication in a large-animal model comparing graft implanted against muscle fascia and periosteum. METHODS: Twenty skeletally mature domestic sheep were implanted with rectangular chambers containing equal weights of morcellized bone graft. Two chambers were implanted into each sheep, one with the open face apposed to the cambium layer of the rib periosteum and the other with the open face apposed to the fascia of the latissimus dorsi muscle. Animals were euthanized at 3, 6, 9, 12, and 24 weeks and the chambers were harvested. Tissue inside the chambers was analyzed for shape conformation to chamber geometry, gross tissue volume, and bone histomorphology. RESULTS: There were no differences in volume or shape of tissue formed in the chambers. However, chambers in contact with fascia consisted almost entirely of fibrovascular tissue, with progressive resorption of the morcellized bone graft and little evidence of new bone. Chambers in contact with periosteum showed active endochondral, direct, and appositional bone formation over time, with increasing calcified tissue area and new bone formation. CONCLUSIONS: Both periosteum and muscle fascia were able to vascularize bone grafts, but bone formation was higher in the periosteum. The periosteum appears to be a more suitable foundation from which to promote flap prefabrication.

Preparation and assessment of glutaraldehyde-crosslinked collagen-chitosan hydrogels for adipose tissue engineering.

A portfolio of crosslinked chitosan:collagen blends was prepared, and their microarchitecture and water binding capacity were studied to investigate their application for adipose tissue engineering. Glutaraldehyde (GA) concentration had little effect on scaffold morphology or water binding capacity. However, the processing freezing temperature prior to lyophilization affected both. In vitro cytocompatibility of pre-adipocytes (PAs) was assessed for a candidate collagen:chitosan blend using two assays. Results confirm the viability of PAs on GA-crosslinked collagen:chitosan scaffolds. A rat subcutaneous pocket assay was employed to assess PA-seeded scaffolds in vivo. Animal tests proved that PA-seeded scaffolds were biocompatible, could induce vascularization, and form adipose tissue.

Three-dimensional adipose tissue model using low shear bioreactors.

Presented here are techniques developed to culture and analyze three-dimensional (3-D) adipose-like tissues as a means to bridge the gap between current limitations in culturing preadipocytes (PAs) and that of providing clinically relevant volumes of adipose tissue useful for soft tissue engineering strategies in reconstructive surgery. Pilot studies were performed to determine techniques to visualize and analyze 3-D PA-like tissues as well as to develop successful strategies to culture 3T3-L1 cells in a high aspect ratio vessel rotating-wall bioreactor both with and without microcarriers. Next, a series of cultures were accessed to verify these techniques as well as to compare the culture of the cells with and without microcarriers. Finally, a perfused rotating-wall bioreactor was used to further investigate the nature of the aggregates or tissues being generated. The aggregates that formed in the perfused system were analyzed via histology and in vivo animal studies. PA-like tissues as large as 4-5 mm in diameter without microcarriers that were capable of lipid-loading and composed of viable cells were achieved. We have successfully demonstrated that large tissue aggregates can be grown in bioreactor culture systems.

Poly(ethylene glycol) hydrogel system supports preadipocyte viability, adhesion, and proliferation.

The ultimate goal of this research is to develop an injectable cell-scaffold system capable of permitting adipogenesis to abrogate soft tissue deficiencies resulting from trauma, tumor resection, and congenital abnormalities. The present work compares the efficacy of photopolymerizable poly(ethylene glycol) and specific derivatives as a scaffold for preadipocyte (adipocyte precursor cell) viability, adhesion, and proliferation. Four variations of a poly(ethylene glycol) scaffold are prepared and examined. The first scaffold consists of poly(ethylene glycol) diacrylate, which is not susceptible to hydrolysis or enzymatic degradation. Preadipocyte death is observed over 1 week in this hydrogel configuration. Adhesion sites, specifically the laminin-binding peptide sequence YIGSR, were incorporated into the second scaffold to promote cellular adhesion as a prerequisite for preadipocyte proliferation. Preadipocytes remain viable in this scaffold system, but do not proliferate in this nondegradable hydrogel. The third scaffold system studied consists of poly(ethylene glycol) modified with the peptide sequence LGPA to permit polymer degradation by cell-secreted collagenase. No adhesion peptide is incorporated into this scaffold system. Cellular proliferation is initially observed, followed by cell death. The previous three scaffold configurations do not permit preadipocyte adhesion and proliferation. In contrast, the fourth system studied, poly(ethylene glycol) modified to incorporate both LGPA and YIGSR, permits preadipocyte adherence and proliferation subsequent to polymer degradation. Our results indicate that a scaffold system containing specific degradation sites and cell adhesion ligands permits cells to adhere and proliferate, thus providing a potential cell-scaffold system for adipogenesis.

Microvascular endothelial cells sustain preadipocyte viability under hypoxic conditions.

Obesity, soft tissue wound healing, adipose tissue engineering, lipomas, and other physiological and pathophysiological conditions necessitate a clear understanding of the interactions between adipocytes and endothelial cells. Adipogenesis and angiogenesis are intimately integrated, despite not being in direct apposition with one another. However, underlying mechanisms have not been elucidated. In this study, the interactions of preadipocytes (PAs) and microvascular endothelial cells are investigated under varying defined O2 conditions, using a coculture system. Results clearly demonstrate that endothelial cells release a soluble factor that sustains PAs viability under hypoxic conditions. Vascular endothelial cell growth factor is not the potential soluble factor (data not shown).

Rheological and recovery properties of poly(ethylene glycol) diacrylate hydrogels and human adipose tissue.

The viscosity and elastic and viscous moduli of poly(ethylene glycol) diacrylate (PEGDA) hydrogels and human abdominal adipose tissue are measured as a function of shear rate and frequency. Results indicate that both materials exhibit shear thinning and are viscoelastic in nature. Rheological tests suggest that the hydrogels become firmer as strain and frequency increase. Adipose tissue, however, begins to fail at higher strains and frequencies. This behavior is confirmed by measuring the complex modulus of both materials as a function of strain. Recovery properties are also measured for each material as a function of deformation. Although PEGDA hydrogels are able to recover up to 78% of their original height after 15% deformation, adipose tissue is not able to recover over the range of deformations tested. The frequencies and strains over which the tests are conducted are those physiologically experienced by the human body. The hydrogels are able to withstand this range of forces and, hence, are appropriate for use as a soft tissue filler material. In addition, the hydrogels swell 38.1% +/- 0.9% independent of surface area. The complex modulus of hydrogels of varying polymer concentrations is also measured as a function of strain to determine the effects of changing polymer content. These results indicate that as polymer content increases, the hydrogels become firmer due to the higher number of polymer chains and behave more elastically.

Ovine model for engineering bone segments.

We propose a large animal model for bone tissue engineering that yields quantitative data and simulates clinical methods and tissue needs. Skeletally mature domestic sheep (n = 20) were each implanted with three rectangular (1 x 1 x 4 cm), hollow tissue-molding chambers that were empty (control) or filled with equal weights (6.71-6.78 g) of particulate autologous bone graft (MBG) or bone graft that was autoclaved to denature stored growth factors (DeMBG). MBG provided scaffold and bioactive factors, and DeMBG provided only scaffold. The chambers were enclosed on five sides and securely implanted so that the open face was apposed to the osteogenic (i.e., cambium) layer of the rib periosteum for 3, 6, 9, 12, or 24 weeks, after which the chambers were harvested and the contents analyzed. Each chamber contained osseous and fibrovascular tissue. MBG-containing chambers had the best maintenance of tissue volume compared with DeMBG-containing or empty chambers, but it still decreased steadily over time. Despite this, the MBG-containing chambers showed continuous active bone formation. There was increasing calcified tissue with penetration of osteogenesis up to a mean of 0.75 +/- 0.15 cm from the periosteum by 9 weeks, and the osteogenic area peaked at 0.59 +/- 0.13 cm2 by 12 weeks. Using quantitative measures that reflect clinical needs (i.e., tissue volume, shape, and quality), it was possible to distinguish differences in performance associated with manipulation of implanted scaffold and bioactive factors. This ovine model may serve as a useful tool to develop clinical osseous tissue-engineering strategies.

A deterministic model of growth factor-induced angiogenesis.

Understanding the formation and structure of a capillary network is critical for any reparative strategy since the capillary network dictates tissue survival, hemodynamics, and mass transport. Vascular assembly and patterning has largely been studied using a reductionist approach where a particular endothelial cell molecular pathway or cellular mechanism is investigated as a relatively closed system. This trend of research has yielded a staggering wealth of genes, proteins, and cells that play critical roles in angiogenesis and some have resulted in successful targeted angiogenic therapies. However, these genes, proteins, and cells do not exist in discrete closed systems, rather they are intimately coupled across spatial and temporal dimensions. Designing experiments to study a single or group of perturbations is fraught with confounding complications. An integrative tool is required that incorporates gene, protein, and cell information and appropriately describes the complex systems behavior of vascular assembly and patterning. In this paper, we propose a new deterministic mathematical formulation to model growth factor-induced angiogenesis. Conductivity of the extracellular matrix for the movement/extension of capillary sprouts is a new concept introduced to account for the heterogeneity and anisotropy of the extracellular matrix. The replacement of traditional endothelial cell density by the capillary indicator function enhances the capabilities of capturing the capillary network sharply in a fine scale (i.e., tracking the dynamics of the tip in uni-cellular scale). Major mechanisms including cell proliferation, sprout branching, and anastomosis are incorporated directly into this continuous mathematical model and model parameters are perturbed to determine the strength of their effect on angiogenesis. The model is fully deterministic and generates the overall dendritic structure of the capillary network morphologically similar to those observed in vivo. The simulations "capture" significant vascular patterning, such as vascular loops and backward growth. Moreover, the simulations provide a deeper understanding of the influence of extracellular matrix on angiogenesis and vascular patterning. An advantage of this model is that the complex physical, chemical and biological processes in angiogenesis can be described and consequently analyzed by a mathematical system with self-contained information.

Three-dimensional, quantitative analysis of desmin and smooth muscle alpha actin expression during angiogenesis.

Angiogenic therapies have been designed for many pathological conditions, but when used as a single therapy, the clinical results have fallen short of expectations. In addition, strategies for vascularizing engineered tissues have been unsuccessful in promoting the formation of an extensive, stable vasculature. Recent evidence suggests that mural cells play a critical role in the success of these approaches, but our current understanding of the function of mural cells in the microvasculature is incomplete. We studied the three-dimensional spatial and temporal kinetics of the mural cell markers desmin and smooth muscle alpha actin during angiogenesis in an in vivo fibrin gel model. The results led to the following conclusions: (1) desmin and smooth muscle alpha actin positive cells are present during the initial development of vessel sprouts; (2) the presence of these cells in the microvasculature is not always an indicator of vessel stability; and (3) the mural cell markers desmin and smooth muscle alpha actin exhibit differential staining patterns during vessel formation. These findings shed new light on the complexity of the relationship between mural cells and the formation of a mature, stable microcirculation.

Breast tissue engineering.

Tissue engineering has the potential to redefine rehabilitation for the breast cancer patient by providing a translatable strategy that restores the postmastectomy breast mound while concomitantly obviating limitations realized with contemporary reconstructive surgery procedures. The engineering design goal is to provide a sufficient volume of viable fat tissue based on a patient's own cells such that deficits in breast volume can be abrogated. To be sure, adipose tissue engineering is in its infancy, but tremendous strides have been made. Numerous studies attest to the feasibility of adipose tissue engineering. The field is now poised to challenge barriers to clinical translation that are germane to most tissue engineering applications, namely scale-up, large animal model development, and vascularization. The innovative and rapid progress of adipose engineering to date, as well as opportunities for its future growth, is presented.

Progress in adipose tissue construct development.

Although the field of tissue engineering has been the focus of a great deal of promise and study, only recently has significant attention been given to the engineering of soft tissues. The applicability of an engineered adipose construct as a basic science model and a reconstructive tool is unquestioned; yet, there have been limitations in previous work, specifically issues of construct size and maintenance over time. This article briefly overviews the pivotal factors necessary for adipocyte growth and differentiation, optimal scaffolds for the engineering of soft tissues, and a means of providing vascular support for these highly demanding cells. Clinical science and bioengineering concepts that may provide the foundation toward the successful in vivo engineering of an adipose tissue construct that maintains its complex three-dimensional shape over time are critically reviewed.

Effects of cryogen spray cooling and high radiant exposures on selective vascular injury during laser irradiation of human skin.

BACKGROUND: Increasing radiant exposure offers a means to increase treatment efficacy during laser-mediated treatment of vascular lesions, such as port-wine stains; however, excessive radiant exposure decreases selective vascular injury due to increased heat generation within the epidermis and collateral damage to perivascular collagen. OBJECTIVE: To determine if cryogen spray cooling could be used to maintain selective vascular injury (ie, prevent epidermal and perivascular collagen damage) when using high radiant exposures (16-30 J/cm2). DESIGN: Observational study. SETTING: Academic hospital and research laboratory. PATIENTS: Twenty women with normal abdominal skin (skin phototypes I-VI). INTERVENTIONS: Skin was irradiated with a pulsed dye laser (wavelength = 585 nm; pulse duration = 1.5 milliseconds; 5-mm-diameter spot) using various radiant exposures (8-30 J/cm2) without and with cryogen spray cooling (50- to 300-millisecond cryogen spurts). MAIN OUTCOME MEASURE: Hematoxylin-eosin-stained histologic sections from each irradiated site were examined for the degree of epidermal damage, maximum depth of red blood cell coagulation, and percentage of vessels containing perivascular collagen coagulation. RESULTS: Long cryogen spurt durations (>200 milliseconds) protected the epidermis in light-skinned individuals (skin phototypes I-IV) at the highest radiant exposure (30 J/cm2); however, epidermal protection could not be achieved in dark-skinned individuals (skin phototypes V-VI) even at the lowest radiant exposure (8 J/cm2). The red blood cell coagulation depth increased with increasing radiant exposure (to >2.5 mm for skin phototypes I-IV and to approximately 1.2 mm for skin phototypes V-VI). In addition, long cryogen spurt durations (>200 milliseconds) prevented perivascular collagen coagulation in all skin types. CONCLUSIONS: Cryogen spurt durations much longer than those currently used in therapy (>200 milliseconds) may be clinically useful for protecting the epidermis and perivascular tissues when using high radiant exposures during cutaneous laser therapies. Additional studies are necessary to prove clinical safety of these protocols.

Integrin-mediated preadipocyte adhesion and migration on laminin-1.

Cell adhesion and migration are key events in many biological processes and depend on extracellular matrix proteins. Understanding these cellular events in adipogenesis is paramount to elucidating the biological mechanisms underlying preadipocyte-specific aspects of obesity, diabetes, and adipose tissue development for the design of pharmaceutical screening and tissue engineering strategies. We quantitatively investigated preadipocyte adhesion and migration on laminin-1 surfaces and identified candidate cognate preadipocyte receptors for laminin-1. In adhesion studies, we found that preadipocytes readily adhered to laminin-1 as compared with other extracellular matrix proteins. In addition, immunocytochemical analysis demonstrated that an array of integrin molecules was present on the surface of preadipocytes. Preadipocyte adhesion on laminin-1 was quantitatively assessed using a sedimentation adhesion assay, and results suggested that preadipocyte adhesion to laminin-1 was mediated by the alpha1beta1 integrin. In addition, digital time-lapse microscopy and quantitative cell tracking revealed that inhibition of the alpha1beta1 integrin resulted in abrogation of preadipocyte migration on laminin-1 surfaces. These results strongly support the hypothesis that preadipocyte adhesion to and migration on laminin-1 substrata are regulated, in part, by integrins.

Automated selection of DAB-labeled tissue for immunohistochemical quantification.

The increased use of immunohistochemistry (IHC) in both clinical and basic research settings has led to the development of techniques for acquiring quantitative information from immunostains. Staining correlates with absolute protein levels and has been investigated as a clinical tool for patient diagnosis and prognosis. For these reasons, automated imaging methods have been developed in an attempt to standardize IHC analysis. We propose a novel imaging technique in which brightfield images of diaminobenzidene (DAB)-labeled antigens are converted to normalized blue images, allowing automated identification of positively stained tissue. A statistical analysis compared our method with seven previously published imaging techniques by measuring each one's agreement with manual analysis by two observers. Eighteen DAB-stained images showing a range of protein levels were used. Accuracy was assessed by calculating the percentage of pixels misclassified using each technique compared with a manual standard. Bland-Altman analysis was then used to show the extent to which misclassification affected staining quantification. Many of the techniques were inconsistent in classifying DAB staining due to background interference, but our method was statistically the most accurate and consistent across all staining levels.

Tissue engineering.

Tissue engineering will potentially change the practice of plastic surgery more than any other clinical specialty. It is an interdisciplinary field that promises new methods of tissue repair. There has been more than $3.5 billion invested in this field since 1990. Relevant areas of progress include advanced computing, biomaterials, cell technology, growth factor fabrication and delivery, and gene manipulation. Beneficial clinical techniques will emerge from continued investigation in each of these areas. Techniques that are developed must be scaled up to industry with products cleared by regulatory agencies and acceptable to clinicians and patients. A goal of tissue engineering is to change clinical practice, yielding improved patient outcomes and lower costs of care.