Tissue engineering and regenerative medicine in musculoskeletal oncology.

Currently used surgical techniques to reconstruct tissue defects after resection of musculoskeletal tumours are associated with high complication rates. This drives a strong demand for innovative therapeutic concepts that are able to improve the clinical outcomes of patients suffering from bone and soft tissue tumours. Tissue engineering and regenerative medicine (TE&RM) provides a technology platform based on biochemical, molecular, cellular and biomaterials modules to selectively direct tissue healing processes for improved defect regeneration. At the same time, precautionary measures have to be taken when these instruments are used in cancer patients to prevent any promotion of tumour growth or metastatic spread. On the other hand, several innovative TE&RM tools are being developed such as multi-functionalized biomaterials, drug-delivering nanomaterials or genetically engineered stem cells that per se have the potential to mediate anti-cancer effects, act synergistically with currently used chemotherapeutics and/or radiotherapy regimens and reduce their side effects. Recently, scientists became conscious that TE&RM strategies may not only be utilized to advance contemporary tissue reconstruction techniques but also to develop personalized diagnostic tools and clinically relevant disease models for cancer patients. Eventually, prospective randomized clinical trials combined with comparative outcome analyses are a conditio sine qua non to shape the benefits of personalized regenerative therapies for the standardized management of patients with musculoskeletal tumours.

A Novel Bioresorbable Implant for Repair of Orbital Floor Fractures.

PURPOSE: To describe clinical, radiologic, and safety outcomes of orbital floor fracture repair using a novel bioresorbable polycaprolactone (PCL) mesh implant (Osteomesh™, Osteopore International, Singapore). METHODS: This is a prospective interventional case series of orbital floor fractures repaired using a novel PCL mesh implant. Clinical evaluation was conducted at presentation and postoperatively at 1, 4, 12, 24 and 48 weeks. Computed tomography (CT) of the orbits was performed 1 year postoperatively. RESULTS: A total of 20 patients were recruited. Mean follow up was 50.4 ± 31.88 weeks. The majority of the patients were male (60%) and of Chinese ethnicity (75%), and the mean age was 39.35 (range 13-69) years. The most common mechanism of injury was assault. The average fracture size was 21.9 mm (range 12-32 mm) in the anteroposterior meridian and 18.65 mm (range 6-27 mm) in the horizontal meridian. Fifty percent of the patients were classified as having a large orbital defect (horizontal width ≥20 mm). The binocular single vision (BSV) score improved from 72.1% preoperatively to 90.8% postoperatively (P < 0.05) for 17 patients who had pre and postoperative charts. BSV improvement did not differ significantly between those with large and small orbital fracture sizes. There were features of neobone formation on CT scan performed 1.5 years after implantation. CONCLUSION: This bioresorbable implant is a promising material for the repair of both small and large orbital floor fractures, giving good functional and aesthetic outcomes.

Inducible hydrogen sulfide synthesis in chondrocytes and mesenchymal progenitor cells: is H2S a novel cytoprotective mediator in the inflamed joint?

Hydrogen sulfide (H(2)S) has recently been proposed as an endogenous mediator of inflammation and is present in human synovial fluid. This study determined whether primary human articular chondrocytes (HACs) and mesenchymal progenitor cells (MPCs) could synthesize H(2)S in response to pro-inflammatory cytokines relevant to human arthropathies, and to determine the cellular responses to endogenous and pharmacological H(2)S. HACs and MPCs were exposed to IL-1ß, IL-6, TNF-α and lipopolysaccharide (LPS). The expression and enzymatic activity of the H(2)S synthesizing enzymes cystathionine-ß-synthase (CBS) and cystathionine-γ-lyase (CSE) were determined by Western blot and zinc-trap spectrophotometry, respectively. Cellular oxidative stress was induced by H(2)O(2), the peroxynitrite donor SIN-1 and 4-hydroxynonenal (4-HNE). Cell death was assessed by 3-(4,5-dimethyl-2-yl)-2,5-diphenyltetrazolium bromide (MTT) and lactate dehydrogenase (LDH) assays. Mitochondrial membrane potential (DCm) was determined in situ by flow cytometry. Endogenous H(2) S synthesis was inhibited by siRNA-mediated knockdown of CSE and CBS and pharmacological inhibitors D,L-propargylglycine and aminoxyacetate, respectively. Exogenous H(2)S was generated using GYY4137. Under basal conditions HACs and MPCs expressed CBS and CSE and synthesized H(2)S in a CBS-dependent manner, whereas CSE expression and activity was induced by treatment of cells with IL-1ß, TNF-α, IL-6 or LPS. Oxidative stress-induced cell death was significantly inhibited by GYY4137 treatment but increased by pharmacological inhibition of H(2)S synthesis or by CBS/CSE-siRNA treatment. These data suggest CSE is an inducible source of H(2)S in cultured HACs and MPCs. H(2)S may represent a novel endogenous mechanism of cytoprotection in the inflamed joint, suggesting a potential opportunity for therapeutic intervention.

Regenerative medicine: implications for craniofacial surgery.

Craniofacial reconstruction of cases with complex anatomy challenges surgeons. The recently emerging field of tissue engineering and regenerative medicine has resulted in a variety of novel therapeutic concepts particularly in the craniofacial area. However, researchers still face significant problems when translating scientific concepts from the bench to the bedside. Reconstruction procedures depend on sustainability, aesthetic outcome, and functionality. Tissue engineering approaches yield powerful tools for long-term satisfying results enabling customized reconstruction and supporting natural healing processes. In conclusion, further advances of tissue-engineered reconstruction need multidisciplinary research to create complex tissue structures and make satisfactory outcomes clinically achievable for most patients. This review highlights clinical advances in the field and gives an overview about current scientific concepts.

Tuberculous extensor tenosynovitis of the hand.

Tuberculous tenosynovitis is a rare manifestation of extrapulmonary tuberculosis (Tb), especially if solely the dorsal hand compartment is affected. In this report, we present the medical history of an immuno-competent 32-year-old man presented with a painful swelling of the right dorsal wrist. Initial inflammation onset had occurred 6 months before he consulted our service, resulting in consultation of several physicians and extensive diagnostic procedures without gaining a specific diagnosis. Finally, after extensive diagnostic tests, a tenosynovectomy was performed and tuberculosis-induced extensor tenosynovitis was detected. The diagnosis was established by positive histology, repeated specific PCR and T-SPOT.TB. Tuberculous tenosynovitis can easily be overlooked as a cause of chronic tenosynovitis particularly in immunocompetent young people lacking any risk factors.

Superior osteogenic capacity for bone tissue engineering of fetal compared with perinatal and adult mesenchymal stem cells.

Mesenchymal stem cells (MSCs) from human adult bone marrow (haMSCs) represent a promising source for bone tissue engineering. However, their low frequencies and limited proliferation restrict their clinical utility. Alternative postnatal, perinatal, and fetal sources of MSCs appear to have different osteogenic capacities, but have not been systematically compared with haMSCs. We investigated the proliferative and osteogenic potential of MSCs from human fetal bone marrow (hfMSCs), human umbilical cord (hUCMSCs), and human adult adipose tissue (hATMSCs), and haMSCs, both in monolayer cultures and after loading into three-dimensional polycaprolactone-tricalcium-phosphate scaffolds.Although all MSCs had comparable immunophenotypes, only hfMSCs and hUCMSCs were positive for the embryonic pluripotency markers Oct-4 and Nanog. hfMSCs expressed the lowest HLA-I level (55% versus 95%-99%) and the highest Stro-1 level (51% versus 10%-27%), and had the greatest colony-forming unit-fibroblast capacity (1.6x-2.0x; p < .01) and fastest doubling time (32 versus 54-111 hours; p < .01). hfMSCs had the greatest osteogenic capacity, as assessed by von-Kossa staining, alkaline phosphatase activity (5.1x-12.4x; p < .01), calcium deposition (1.6x-2.7x in monolayer and 1.6x-5.0x in scaffold culture; p < .01), calcium visualized on micro-computed tomography (3.9x17.6x; p < .01) and scanning electron microscopy, and osteogenic gene induction. Two months after implantation of cellular scaffolds in immunodeficient mice, hfMSCs resulted in the most robust mineralization (1.8x-13.3x; p < .01).The ontological and anatomical origins of MSCs have profound influences on the proliferative and osteogenic capacity of MSCs. hfMSCs had the most proliferative and osteogenic capacity of the MSC sources, as well as being the least immunogenic, suggesting they are superior candidates for bone tissue engineering.

The pro-inflammatory oxidant hypochlorous acid induces Bax-dependent mitochondrial permeabilisation and cell death through AIF-/EndoG-dependent pathways.

At sites of chronic inflammation, such as in the inflamed rheumatoid joint, activated neutrophils release hydrogen peroxide (H(2)O(2)) and the enzyme myeloperoxidase to catalyse the formation of hypochlorous acid (HOCl). 3-chlorotyrosine, a marker of HOCl in vivo, has been observed in synovial fluid proteins from rheumatoid arthritis patients. However the mechanisms of HOCl-induced cytotxicity are unknown. We determined the molecular mechanisms by which HOCl induced cell death in human mesenchymal progenitor cells (MPCs) differentiated into a chondrocytic phenotype as a model of human cartilage cells and show that HOCl induced rapid Bax conformational change, mitochondrial permeability and release of intra-mitochondrial pro-apoptotic proteins which resulted in nuclear translocation of AIF and EndoG. siRNA-mediated knockdown of Bax substantially prevented mitochondrial permeability, release of intra-mitochondrial pro-apoptotic proteins. Cell death was inhibited by siRNA-mediated knockdown of Bax, AIF or EndoG. Although we observed several biochemical markers of apoptosis, caspase activation was not detected either by western blotting, fluorescence activity assays or by using caspase inhibitors to inhibit cell death. This was further supported by findings that (1) in vitro exposure of recombinant human caspases to HOCl caused significant inhibition of caspase activity and (2) the addition of HOCl to staurosporine-treated MPCs inhibited the activity of cellular caspases. Our results show for the first time that HOCl induced Bax-dependent mitochondrial permeability which led to cell death without caspase activity by processes involving AIF/EndoG-dependent pathways. Our study provides a novel insight into the potential mechanisms of cell death in the inflamed human joint.

Non-linear optical microscopy and histological analysis of collagen, elastin and lysyl oxidase expression in breast capsular contracture.

BACKGROUND: Capsular contracture is one of the most common complications in surgical interventions for aesthetic breast augmentation or post-mastectomy breast reconstruction involving the use of silicone prostheses. Although the precise cause of capsular contracture is yet unknown, the leading hypothesis is that it is caused by long-term unresolved foreign body reaction towards the silicone breast implant. To authors' best knowledge, this is the first study that elucidates the presence of lysyl oxidase (LOX)-an enzyme that is involved in collagen and elastin crosslinking within fibrous capsules harvested from patients with severe capsular contracture. It was hypothesized that over-expression of LOX plays a role in the irreversible crosslinking of collagen and elastin which, in turn, stabilizes the fibrous proteins and contributes to the progression of capsular contracture. METHODS: Eight fibrous capsules were collected from patients undergoing capsulectomy procedure, biomechanical testing was performed for compressive Young's moduli and evaluated for Type I and II collagen, elastin and LOX by means of non-linear optical microscopy and immunohistology techniques. RESULTS: Observations revealed the heterogeneity of tissue structure within and among the collected fibrous capsules. Regardless of the tissue structure, it has been shown that LOX expression was intensified at the implant-to-tissue interface. CONCLUSION: Our results indicate the involvement of LOX in the initiation of fibrous capsule formation which ultimately contributes towards the progression of capsular contracture.

Cell guidance in tissue engineering: SDF-1 mediates site-directed homing of mesenchymal stem cells within three-dimensional polycaprolactone scaffolds.

Cell guidance is a new tissue engineering concept aimed at total in vivo tissue engineering without the need for cell seeding. This technique aims to create a biomimetic environment through constant delivery of cytokines to different areas of an implanted scaffold, such that site-specific homing of cells can be achieved. In this study, expression of CXCR4 on mesenchymal stem cells (MSCs) was characterized by immunohistochemistry and flow cytometry, subsequent to which chemotaxis toward stromal cell-derived factor 1 (SDF-1) was demonstrated. In a subsequent three-dimensional in vitro study, MSCs were shown to migrate within a polycaprolactone scaffold in response to SDF-1, such that polarized tissue formation could be achieved. A customized cytokine microdelivery system comprising a reservoir housing system and microneedle apparatus was fabricated to ensure constant delivery of SDF-1 to the scaffold. Following on this experiment, we demonstrated in an in vivo rat bone tissue engineering model that a cytokine combination consisting of vascular endothelial growth factor, SDF-1, and bone morphogenetic protein-6 delivered at 10-day intervals through the microneedle apparatus could lead to tissue formation through migrating cell fronts, with evidence of angiogenesis and vascularization without the need for cell seeding on scaffolds prior to implantation. In summary, cell guidance offers an advancement in cellular methodology for tissue engineering, and promises a novel, minimally invasive option for tissue regeneration.

Cartilage engineering in reconstructive surgery: auricular, nasal and tracheal engineering from a surgical perspective.

This review provides an update on cartilage tissue engineering with particular focus on the head and neck. It is aimed at scientists and clinicians who are interested in tissue engineering and its clinical applicability. Principal tissue engineering strategies are summarized in the first part of this review. In the second part, current clinical approaches to auricular, nasal and tracheal reconstruction are discussed from a surgical perspective. By this approach, the requirements for clinical applicability are outlined and new insight into relevant aims of research is given to accelerate the transfer from bench to bedside.

Microneedle physical contact as a therapeutic for abnormal scars.

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.

[Soft tissue fillers: state of the art and future perspectives]. / Injizierbare Füllmaterialien ­ Update und Zukunftsperspektive.

In addition to cosmetic use, filler materials are frequently employed for reconstruction of contour defects. Scars and tissue defects after accidents, surgery and irradiation are also ideal indications for injectables. The ideal filler should be easy to inject, easy to form, and should remain in the body as long as possible. It should neither be allergenic or carcinogenic nor susceptible to infections or biofilms, but still potentially reversible, temperature-stable, and cheap. There is still no augmentation material that fulfils all of these criteria. A wide range of materials with widely varying characteristics is available on the market. Hyaluronic acid, collagen (human or bovine), polylactic acid (PLLA), calcium hydroxyapatite (CaHA), polymethylmethacrylate (PMMA), silicone and natural fat are all used in clinical practice for various indications. The current research efforts carried out at industrial institutions and universities are directed towards the development of augmentation materials with higher biocompatibility on the one hand and better durability on the other. In this review we provide an overview on filler materials currently used with their pros and cons. We further give an outlook on promising new approaches in the development pipeline.

Donor-dependent variances of human adipose-derived stem cells in respect to the in-vitro endothelial cell differentiation capability.

Human adipose-derived stem cells (ASC) have been shown to differentiate into mature adipocytes and to play an important role in creating the vasculature, necessary for white adipose tissue to function. To study the stimulatory capacity of ASC on endothelial progenitor cells we used a commercially available co-culture system (V2a - assay). ASC, isolated from lipoaspirates of 18 healthy patients, were co-cultured for 13 d on endothelial progenitor cells. Using anti CD31 immunostaining, cells that had undergone endothelial differentiation were quantified after the defined co-cultivation period. Endothelial cell differentiation was observed and demonstrated by an increase in area covered by CD31+ cells compared with less to no endothelial cell differentiation in negative and media-only controls. Enzyme-linked immunosorbent assay (ELISA) for vascular endothelial growth factor (VEGF) in supernatant medium collected during the co-cultivation period revealed elevated VEGF levels in the co-culture samples as compared with ASC cultures alone, whereas no increase in adiponectin was detected by ELISA. These findings help to provide further insights in the complex interplay of adipose derived cells and endothelial cells and to better understand the diversity of ASCs in respect of their stimulatory capacity to promote angiogenesis in vitro.

Breast Augmentation and Reconstruction from a Regenerative Medicine Point of View: State of the Art and Future Perspectives.

Breast reconstruction and augmentation are very common procedures, yet the prevailing current methods utilize silicone implants that may have significant local complications requiring reoperation. Lipofillling is increasingly used to contour and is considered safe, however, its utility is limited by significant volume loss. A new approach could offer an alternative and increase the scope of patient choice. A small number of teams around the world are investigating a breast tissue engineering (TE) paradigm. Conventional breast TE concepts are based on seeding a scaffold with the patients' own stem cells. However, the clinical viability of many of these approaches is limited by their costs in relevant volumes. In this article the state of the art of tissue-engineered breast reconstruction is reviewed and future perspectives are presented and discussed.

Transformation of Breast Reconstruction via Additive Biomanufacturing.

Adipose tissue engineering offers a promising alternative to current breast reconstruction options. However, the conventional approach of using a scaffold in combination with adipose-derived precursor cells poses several problems in terms of scalability and hence clinical feasibility. Following the body-as-a-bioreactor approach, this study proposes a unique concept of delayed fat injection into an additive biomanufactured and custom-made scaffold. Three study groups were evaluated: Empty scaffold, Scaffold containing 4 cm(3) lipoaspirate and Empty scaffold +2-week prevascularisation period. In group 3, of prevascularisation, 4 cm(3) of lipoaspirate was injected into scaffolds after 2 weeks. Using a well-characterised additive biomanufacturing technology platform, patient-specific scaffolds made of medical-grade-polycaprolactone were designed and fabricated. Scaffolds were implanted in subglandular pockets in immunocompetent minipigs (n = 4) for 24-weeks. Angiogenesis and adipose tissue regeneration were observed in all constructs. Histological evaluation showed that the prevascularisation + lipoaspirate group had the highest relative area of adipose tissue (47.32% ± 4.12) which was significantly higher than both lipoaspirate-only (39.67% ± 2.04) and empty control group (8.31% ± 8.94) and similar to native breast tissue (44.97% ± 14.12). This large preclinical animal study provides proof-of-principle that the clinically applicable prevascularisation and delayed fat-injection techniques can be used for regeneration of large volumes of adipose tissue.

Polylactides in additive biomanufacturing.

New advanced manufacturing technologies under the alias of additive biomanufacturing allow the design and fabrication of a range of products from pre-operative models, cutting guides and medical devices to scaffolds. The process of printing in 3 dimensions of cells, extracellular matrix (ECM) and biomaterials (bioinks, powders, etc.) to generate in vitro and/or in vivo tissue analogue structures has been termed bioprinting. To further advance in additive biomanufacturing, there are many aspects that we can learn from the wider additive manufacturing (AM) industry, which have progressed tremendously since its introduction into the manufacturing sector. First, this review gives an overview of additive manufacturing and both industry and academia efforts in addressing specific challenges in the AM technologies to drive toward AM-enabled industrial revolution. After which, considerations of poly(lactides) as a biomaterial in additive biomanufacturing are discussed. Challenges in wider additive biomanufacturing field are discussed in terms of (a) biomaterials; (b) computer-aided design, engineering and manufacturing; (c) AM and additive biomanufacturing printers hardware; and (d) system integration. Finally, the outlook for additive biomanufacturing was discussed.

Peroxynitrite mediates calcium-dependent mitochondrial dysfunction and cell death via activation of calpains.

Chondrocyte cell death is a hallmark of inflammatory and degenerative joint diseases such as rheumatoid arthritis (RA) and osteoarthritis (OA), but the molecular and cellular mechanisms involved have yet to be elucidated. Because 3-nitrotyrosine, a marker for reactive nitrogen species such as peroxynitrite, has been observed in OA and RA cartilage and has been associated with chondrocyte cell death, we investigated the mechanisms by which peroxynitrite induces cell death in human articular chondrocytes. The earliest biochemical event observed, subsequent to treatment with either peroxynitrite or the peroxynitrite generator SIN-1, was a rapid rise in intracellular calcium that lead to mitochondrial dysfunction and cell death. Although, chondrocyte death exhibited several classical hallmarks of apoptosis, including annexin V labeling, increased fraction of cells with subG1 DNA content and DNA condensation, we did not find evidence for caspase involvement either by Western blotting, fluorimetric assays, or caspase inhibition. Additionally, peroxynitrite did not inhibit cellular caspase activity. Furthermore, using other established assays of cell viability, including the MTT assay and release of lactate dehydrogenase, we found that the predominant mode of cell death involved calcium-dependent cysteine proteases, otherwise known as calpains. Our data show, for the first time, that peroxynitrite induces mitochondrial dysfunction in cells via a calcium-dependent process that leads to caspase-independent apoptosis mediated by calpains.

Additive manufacturing in biomedical sciences and the need for definitions and norms.

The application of additive biomanufacturing represents one of the most rapidly advancing areas of biomedical science, in which engineers, scientists, and clinicians are contributing to the future of health care. The combined efforts of a large number of groups around the globe have developed a strong research thrust that has resulted in a large number of publications. Reviewing this body of literature, there is an increasing trend of research groups inventing their own definitions and terminology. This has made it difficult to find and compare the results. Therefore, to move the field constructively forward, it is a conditio sine qua non to clarify various terminologies and standards. Based on this background, this article advocates tightening the terminology and has the objective of penning out definitions that will ultimately allow the development of official industry standard terms, such as American Society for Testing and Materials and or International Organization for Standardization for technologies developed for Tissue Engineering and Regenerative Medicine.

Repair of calvarial defects with customized tissue-engineered bone grafts I. Evaluation of osteogenesis in a three-dimensional culture system.

Bone generation by autogenous cell transplantation in combination with a biodegradable scaffold is one of the most promising techniques being developed in craniofacial surgery. The objective of this combined in vitro and in vivo study was to evaluate the morphology and osteogenic differentiation of bone marrow derived mesenchymal progenitor cells and calvarial osteoblasts in a two-dimensional (2-D) and three-dimensional (3-D) culture environment (Part I of this study) and their potential in combination with a biodegradable scaffold to reconstruct critical-size calvarial defects in an autologous animal model [Part II of this study; see Schantz, J.T., et al. Tissue Eng. 2003;9(Suppl. 1):S-127-S-139; this issue]. New Zealand White rabbits were used to isolate osteoblasts from calvarial bone chips and bone marrow stromal cells from iliac crest bone marrow aspirates. Multilineage differentiation potential was evaluated in a 2-D culture setting. After amplification, the cells were seeded within a fibrin matrix into a 3-D polycaprolactone (PCL) scaffold system. The constructs were cultured for up to 3 weeks in vitro and assayed for cell attachment and proliferation using phase-contrast light, confocal laser, and scanning electron microscopy and the MTS cell metabolic assay. Osteogenic differentiation was analyzed by determining the expression of alkaline phosphatase (ALP) and osteocalcin. The bone marrow-derived progenitor cells demonstrated the potential to be induced to the osteogenic, adipogenic, and chondrogenic pathways. In a 3-D environment, cell-seeded PCL scaffolds evaluated by confocal laser microscopy revealed continuous cell proliferation and homogeneous cell distribution within the PCL scaffolds. On osteogenic induction mesenchymal progenitor cells (12 U/L) produce significantly higher (p < 0.05) ALP activity than do osteoblasts (2 U/L); however, no significant differences were found in osteocalcin expression. In conclusion, this study showed that the combination of a mechanically stable synthetic framework (PCL scaffolds) and a biomimetic hydrogel (fibrin glue) provides a potential matrix for bone tissue-engineering applications. Comparison of osteogenic differentiation between the two mesenchymal cell sources revealed a similar pattern.

Repair of calvarial defects with customised tissue-engineered bone grafts II. Evaluation of cellular efficiency and efficacy in vivo.

We have demonstrated in Part I of this study [see Schantz, J.-T., et al., Tissue Eng. 2003;9(Suppl. 1): S-113-S-126; this issue] that bone marrow-derived progenitor cells and calvarial osteoblasts could be successfully directed into the osteogenic lineage and cultured in three-dimensional (3-D) polycaprolactone (PCL) scaffolds. The objective of the second part of the study was to evaluate and to compare tissue engineered cell-polymer constructs using calvarial osteoblasts (group I) and mesenchymal progenitor cells (MPCs; group II) for the reconstruction of critical-size and three-dimensionally complex cranial defects. In 30 New Zealand White rabbits, bilateral parietal critical-size defects were created. On the basis of computed tomography scans, customized PCL scaffolds with precisely controlled microarchitecture were fabricated, using a rapid prototyping technology. Bone marrow-derived progenitor cells and osteoblasts were isolated and expanded in culture. Osteoblasts (group I) and mesenchymal progenitor cells (group II) were seeded in combination with a fibrin glue suspension into 40 PCL scaffolds. After incubating for 3 days in static culture, the PCL scaffold-cell constructs as well as nonseeded PCL scaffolds (control group) were implanted into 15-mm-diameter calvarial defects. Reconstruction of the cranium and bone formation were evaluated after 3 months. In vivo results indicated osseous tissue integration within the implant and functionally stable restoration of the calvarium. Islands of early bone formation could be observed in X-ray radiographs and in histological sections. Implants showed a high cell:ECM ratio and a dense vascular network. Mechanical testing of the reconstructed area revealed partial integration with the surrounding corticocancellous calvarial bone. The amount (area) of calcification, measured by clinical computed tomography, indicated that cell-seeded constructs measured about 60% more than unrepaired or unseeded scaffolds. Mechanical investigations revealed that stiffness reached 52 +/- 29 and 44 +/- 16 MPa for MPC- and osteoblast-seeded scaffolds, respectively. The yield strength for the push-out tests was 180 +/- 36 N for normal calvarial bone, 90 +/- 1 N for unrepaired site, and 106 +/- 10 N for unseeded constructs, which is about 60% of normal bone strength. MPC- and osteoblast-seeded scaffolds indicated a yield strength of 149 +/- 15 and 164 +/- 42 N, respectively, which is about 85-90% of normal bone. This study demonstrated that customized biodegradable polymeric implants may be used to deliver osteogenic cells and enhance bone formation within critically-sized defects in vivo. The use of rapid prototyping technology to produce scaffolds with controlled external geometry and microarchitecture offers new possibilities in the functional and aesthetic reconstruction of complex craniofacial defects.