Vascular Guidance: Microstructural Scaffold Patterning for Inductive Neovascularization

Current tissue engineering techniques are limited by inadequate vascularisation and perfusion of cell-scaffold constructs. Microstructural patterning through biomimetic vascular channels within a polymer scaffold might induce neovascularization, allowing fabrication of large engineered constructs. The network of vascular channels within a frontal-parietal defect in a patient, originating from the anterior branch of the middle meningeal artery, was modeled using computer-aided design (CAD) techniques and subsequently incorporated into polycaprolactone (PCL) scaffolds fabricated using fused deposition modeling (FDM). Bone marrow-derived mesenchymal stem cells (MSCs) were seeded onto the scaffolds and implanted into a rat model, with an arteriovenous bundle inserted at the proximal extent of the vascular network. After 3 weeks, scaffolds were elevated as a prefabricated composite tissue-polymer flap and transferred using microsurgical technique. Histological examination of explanted scaffolds revealed vascular ingrowth along patterned channels, with abundant capillary and connective tissue formation throughout experimental scaffolds, while control scaffolds showed only granulation tissue. All prefabricated constructs transferred as free flaps survived and were viable. We term this concept “vascular guidance,” whereby neovascularization is guided through customized channels in a scaffold. Our technique might potentially allow fabrication of much larger tissue-engineered constructs than current technologies allow, as well as allowing tailored construct fabrication with a patient-specific vessel network based on CT scan data and CAD technology

Experimental and clinical evaluation of tissue engineered bone grafts for calvarial reconstrution

Ph.DDOCTOR OF PHILOSOPH

Human circulating peripheral blood mononuclear cells for calvarial bone tissue engineering

Large-scale production of engineered tissues requires an adequate source of expandable cells. Current strategies that involve harvesting of cells from donor tissue or bone marrow for tissue engineering are invasive and unfeasible for obtaining large quantities of cells in a clinical setting. Peripheral blood has been reported to contain circulating hematopoietic cells as well as, in significantly smaller quantities, mesenchymal cells An adherent subset of CD14+ mononuclear cells was isolated from human peripheral venous blood and characterized in vitro by light microscopy, immunohistochemistry, flow cytometry, and quantitative differentiation assays. These cells were then evaluated for the purposes of tissue engineering in a rat calvarial defect model, in combination with biodegradable polymer matrices made from poly-e-caprolactone. Specimens were analyzed 6 weeks after implantation with histologic analysis, microcomputed tomography, and HLA immunostaining. CD14+ mononuclear cells were induced to differentiate into osteoblast-like cells in vitro, with areas of mineralization. In a rat calvarial defect model, tissue-engineered bone with evidence of mineralization was formed within 6 weeks. HLA immunohistochemistry demonstrated that de novo bone formation originated from the transplanted human cells. These findings show, for the first time, to our knowledge, the derivation of bone from human blood. They also demonstrate the utility of circulating mononuclear cells as a minimally invasive, potentially unlimited pool of cells for tissue engineering and organ regeneration

Computer aided design of scaffolds for bone tissue engineering [Regeneration von Knochendefekten mit computergesteuerter Herstellung von Gerusttragern]

Large bone defects resulting from trauma or tumour surgery are still considered a major challenge in clinical practice. Despite high clinical demand, current treatment options have a number of shortcomings. Bone tissue engineering (BTE)-strategies have therefore been extensively investigated in recent years. The invention of additive manufacturing (AM)-techniques two decades ago has had a huge impact on the BTE field ever since then: Via AM a solid three dimensional structure can be formed from a digital 3D model using a layer-by-layer fabrication process. In the beginning, AM was mainly used to build 3D models of bone pathologies (e. g. fractures, bone tumours) to enable haptic assessment before and during surgery for planning and executing the surgical procedure. However, as new techniques and materials have been developed, AM can nowadays be used to manufacture ultrastructured three dimensional scaffolds for BTE applications as well. Providing control over the internal scaffold architecture on micrometer scale as well as over the external macroscopic scaffold shape, AM enables the fabrication of patientspecific and/or custom-made scaffolds individually tailored to exactly match the size and requirements (e. g. mechanical properties) of a bone defect. In the future, new technologies that enable the direct fabrication of scaffolds with a parallel spatially controlled deposition of cells and growth factors will further underpin the clinical application of bone tissue engineering

Beyond the vernacular: new sources of cells for bone tissue engineering

Recent developments in stem cell biology have led to the discovery of new sources of adult stem cells with potential for osteogenic differentiation. In this article, the authors review the active field of research into new cell sources that are being investigated for use in bone tissue engineering. These include adipogenic, muscle, intraoral, dermal, and peripheral blood stem cells. The concept of "cell guidance," where cells are induced to home into a scaffold without the need for prior cell seeding, is also discussed. These new cell sources have the advantages of decreased morbidity during harvesting from patients and increased availability compared with traditional sources of cells for bone tissue engineering, such as end organ-derived osteoblasts, bone marrow mesenchymal stem cells, and periosteal progenitor cells. The move beyond common sources of cells is perhaps the single most important recent development in bone tissue engineering research

Comparison of the differentiation potential of cell populations isolated from human lipoaspirate, trabecular bone tissue and periosteal tissue

Recent studies indicate that a variety of cells derived from adult human tissues show the capacity to change their tissue specific differentiation program. In this study we harvested cells from lipoaspirated adipose tissue, trabecular bone and pericranium tissue in order to evaluate and compare the differentiation potential of these populations by exposing them to adipogenic, chondrogenic, osteogenic and neurogenic induction media. Histological and immunochemistry analysis after adipogenic induction revealed adipocyte-like characteristic for all populations. Under osteogenic induction, all populations expressed the bone matrix proteins osteocalcin, osteopontin, osteonectin and secreted alkaline phosphatase, an early bone marker, into the culture medium. The chondrogenic potential was evaluated after staining of acidic glycoconjugates within the extracellular matrix. All induced cell populations were stained positive, indicating the presence of de novo cartilage. After neuronal induction, responsive cells exhibited neuron-like morphology with refractile cell bodies, extended long processes terminating in typical growth cones and long axon-like filopodia. Immunocytochemistry revealed the expression of neurofilament M and neuron specific enolase. Due to the use of heterogeneous cell populations each cell population preferentially expressed the phenotype of the tissue it was isolated from. In comparison; under the respective experimental conditions the periosteal and adipose derived cell populations demonstrated equal versatile developmental potentials according to cell morphology, immunocytochemistry and histology. In contrast the trabecular derived cells displayed a lower differentiation potential. These findings indicate that there are subpopulations within the isolated cell populations allowing all these cell preparations to express adipocyte-, osteoblast-, chondrocyte-, and neuron-like phenotypes.</p

Microneedle physical contact as a therapeutic for abnormal scars

Abstract Background Abnormal (keloid and hypertrophic) scars are a significant affliction with no satisfactory single modality therapy to-date. Available options are often ineffective, painful, potentially hazardous, and require healthcare personnel involvement. Herein a self-administered microneedle device based on drug-free physical contact for inhibiting abnormal scars is reported. Its therapeutic activity through microneedle contact eliminates hazards associated with toxic anti-scarring drugs while self-treatment enables administration flexibility. Methods The microneedle patch was fabricated with FDA-approved liquid crystalline polymer under good manufacturing practice. It was first tested to ascertain its ability to inhibit (keloid) fibroblast proliferation. Later the microneedle patch was examined on the rabbit ear hypertrophic scar model to explore its potential in inhibiting the generation of abnormal scars post-injury. Finally, the microneedle patch was applied to the caudal region of a hypertrophic scar located on a female patient’s dorsum to verify clinical efficacy. Results On untreated control cultures, barely any non-viable fibroblasts could be seen. After 12-h treatment with the microneedle patch, the non-viable proportion increased to 83.8 ± 11.96%. In rabbit ear hypertrophic scar model, 100% of the control wounds without the presence of patches on rabbit ears generated regions of raised dermis originating from the wound site (3/3), whereas microneedle treatment prevented dermis tissue thickening in 83.33% of the wounds (15/18). In the clinical test, the microneedle patch was well tolerated by the patient. Compared to the untreated region, microneedle treatment decreased the number of infiltrated inflammatory cells, with less disrupted dermis tissue architecture and more flattened appearance. Conclusions A self-administered, drug-free microneedle patch appears highly promising in reducing abnormal scarring as observed from in vitro, in vivo and clinical experiments. Larger cohort clinical studies need to be performed to validate its efficacy on abnormal scars

Transformation of Breast Reconstruction via Additive Biomanufacturing

Adipose tissue engineering offers a promising alternative to current breast reconstruction options. \ud However, the conventional approach of using a scaffold in combination with adipose-derived precursor \ud cells poses several problems in terms of scalability and hence clinical feasibility. Following the body-as-\ud a-bioreactor approach, this study proposes a unique concept of delayed fat injection into an additive \ud biomanufactured and custom-made scaffold

Evaluation of polycaprolactone scaffold degradation for 6 months in vitro and in vivo

The use of polycaprolactone (PCL) as a biomaterial, especially in the fields of drug delivery and tissue engineering, has enjoyed significant growth. Understanding how such a device or scaffold eventually degrades in vivo is paramount as the defect site regenerates and remodels. Degradation studies of three-dimensional PCL and PCL-based composite scaffolds were conducted in vitro (in phosphate buffered saline) and in vivo (rabbit model). Results up to 6 months are reported. All samples recorded virtually no molecular weight changes after 6 months, with a maximum mass loss of only about 7% from the PCL-composite scaffolds degraded in vivo, and a minimum of 1% from PCL scaffolds. Overall, crystallinity increased slightly because of the effects of polymer recrystallization. This was also a contributory factor for the observed stiffness increment in some of the samples, while only the PCL-composite scaffold registered a decrease. Histological examination of the in vivo samples revealed good biocompatibility, with no adverse host tissue reactions up to 6 months. Preliminary results of medical-grade PCL scaffolds, which were implanted for 2 years in a critical-sized rabbit calvarial defect site, are also reported here and support our scaffold design goal for gradual and late molecular weight decreases combined with excellent long-term biocompatibility and bone regeneration. (C) 2008 Wiley Periodicals, Inc. J Biomed Mater Res 90A: 906-919, 200