The combined mechanism of bone morphogenetic protein- and calcium phosphate-induced skeletal tissue formation by human periosteum derived cells.

When combining osteogenic progenitor cells such as human periosteum derived cells (hPDCs) with osteoconductive biomaterials like calcium phosphate (CaP)-scaffolds, in vivo bone formation can be achieved. This process is dependent on the early activation of Bone morphogenetic protein (BMP)-signalling. However, the bone forming process is slow and routinely only a limited amount of bone and bone marrow is formed. Therefore, we hypothesised that a robust clinically relevant outcome could be achieved by adding more physiological levels of potent BMP-ligands to these cell- and CaP-based constructs. For this, hPDCs were characterised for their responsiveness to BMP-ligands upon in vitro 2D stimulation. BMP-2, -4, -6 and -9 robustly induced osteochondrogenic differentiation. Subsequently, these ligands were coated onto clinically approved CaP-scaffolds, BioOss® and CopiOs®, followed by hPDC-seeding. Protein lysates and conditioned media were investigated for activation of BMP signalling pathways. Upon in vivo implantation, the most abundant bone formation was found in BMP-2 and BMP-6-coated scaffolds. Implanted cells actively contributed to the newly formed bone. Remnants of cartilage could be observed in BMP-coated CopiOs®-constructs. Computational analysis displayed that the type of BMP-ligand as well as the CaP-scaffold affects skeletal tissue formation, observed in a qualitative as well as quantitative manner. Furthermore, the in vitro mechanism appears to predict the in vivo outcome. This study presents further evidence for the potential of BMP-technology in the development of clinically relevant cell-based constructs for bone regenerative strategies.

Outcome of Endoprosthesis used in Limb Salvage Surgery in a Malaysian Orthopaedic Oncology Centre.

Introduction: To describe the duration of survival among bone tumour patients with endoprosthesis reconstruction and to determine frequency of implant failure, revision of surgery, and amputation after endoprosthesis reconstruction. Materials and methods: A retrospective cross-sectional review of all patients with either primary bone tumour or secondary bone metastases treated with en bloc resection and endoprosthesis reconstruction from January 2008 to December 2020. Results: A total of 35 failures were recorded among the 27 (48.2%) patients with endoprostheses. Some of the patients suffered from one to three types of modes of failure on different timelines during the course of the disease. Up to eight patients suffered from more than one type of failure throughout the course of the disease. Out of all modes of failure, local recurrence (type 5 failure) was the most common, accounting for 25.0% of all failure cases. Four patients (7.1%) eventually underwent amputation, which were either due to infection (2 patients) or disease progression causing local recurrence (2 patients). Conclusion: The overall result of endoprosthesis reconstruction performed in our centre was compatible with other centres around the world. Moreover, limb salvage surgery should be performed carefully in a selected patient group to maximise the benefits of surgery.

[Differential diagnosis and management of hemangioma at geniculate ganglion].

Objective: To investigate the clinical characteristics, differential diagnosis, treatments and prognosis of facial nerve hemangioma and schwannoma at genicular ganglion, so as to provide reference for clinical diagnosis and treatments of facial nerve tumor at genicular ganglion. Methods: Clinical data of 13 patients with facial nerve tumors at genicular ganglion confirmed by postoperative pathology in the Ninth People's Hospital affiliated to Shanghai Jiaotong University School of Medicine from March 2018 to April 2020 were retrospectively analyzed, including seven cases of hemangioma and six cases of schwannoma. There were eight males and five females. Their ages ranged from 20 to 65, with an average age of 40. The course of disease ranged from 3 to 118 months, with an average of 52 months. All the patients underwent preoperative HRCT of the temporal bone and facial nerve dynamic contrast-enhanced(DCE) MRI examinations. All the patients had detailed surgical procedures and at least one-year postoperative follow-up. Results: On HRCT of the temporal bone, (4/7) hemangioma at geniculate ganglion showed characteristic honeycomb appearance, while 6/6 schwannoma and 3/7 hemangiomas showed expansive bone changes. On DCE-MRI, geniculate ganglion hemangioma (7/7) showed characteristic "point-to-surface" enhancement, and schwannoma (6/6) showed characteristic "face-to-surface" enhancement. For five hemangioma-patients with HB-Ⅱ-Ⅳ before surgery, the facial nerve anatomy was completely preserved through transcanal endoscopic approach(TEA), and the facial nerve function improved one year after surgery (two cases of HB-I, two cases of HB-Ⅱ, and one case of HB-Ⅲ). For two patients, with preoperative facial nerve function HB-Ⅴ-Ⅵ, since their tumors was inseparable from the nerves, they were performed with facial nerve anastomosis during the surgery, and the facial nerve function was improved to HB-Ⅳ level one year after surgery. For six patients with meningioma whose facial nerve function was greater than or equal to HB-Ⅲ, based on the preoperative hearing level, the involved segments, and duration of facial paralysis, three of them were conducted surgeries through middle cranial fossa approach, one by translabyrinthine approach, and one via mastoid approach. Two patients among them with complete facial paralysis over three years preoperatively were not performed facial nerve anastomosis after total resections of the tumors, and there was no improvement in facial nerve function one year after surgery. Three patients underwent facial nerve anastomosis after total tumor resections, and their facial nerve function was HB-Ⅲ in one patient, HB-Ⅳ in two patients one year after surgery. One patient (preoperative HB-Ⅲ) had a normal hearing level preoperatively, and the tumor involved the labyrinth segment. To protect the hearing, partial tumor was resected through the middle cranial fossa approach, and facial nerve function improved to HB-Ⅱ one year after surgery. Conclusions: Temporal bone HRCT combined with DCE-MRI are useful for the differential diagnosis of hemangioma and schwannoma at geniculate ganglion and provide references for preoperative clinical decision makings. It is extremely necessary to select the appropriate surgical approach based on the patient's hearing and involved segments. For geniculate ganglion hemangioma, early surgery can improve the possibilities of anatomical integrity of facial nerve, thereby improving facial nerve function postoperatively.TEA is a kind of surgical method worth consideration, with the characteristics of minimally invasive, favorable postoperative features, and so on. For schwannoma, one-stage functional reconstruction of the facial nerve is recommended during the resection of the tumors because of the inevitable damage to the anatomical integrity of the facial nerve.

[Clinical application of endoscope combined with microscope for the microvascular decompression in hemifacial spasm].

Objective: To evaluate the effectiveness and safety of the endoscope combined with microscope for the microvascular decompression in hemifacial spasm. Methods: A total of 26 patients underwent endoscope combined with microscopic facial nerve microvascular decompression through retrolabyrinthine approach from January 2013 to December 2016 were retrospectively reviewed in Ear Institute, Shanghai Jiaotong University School of Medicine. Among them, 9 were male and 17 were female, with a mean age of (51.9±11.4) years;15 cases of left side and 11 of right side patients were followed up for 1-3 years. The pre-and post-operative Cohen Classification was used for hemifacial spasm, House-Brackmann Grade for facial nerve function, hearing level and complication rates were reviewed. SPSS 19.0 software was used to analyze the data. Results: All 26 patients were operated successfully. No recurrence was seen during 1-3 year follow-up. Post-operative Cohen Grade were as follows: 25 cases with Cohen Grade I and 1 case with Cohen Grade II. The difference in Cohen grade between pre-and post-operative was statistically significant (Z=-4.87, P<0.01). Post-operative facial nerve function was satisfactory in all patients (House-Brackmann Grade I-II in all patients). No hearing loss was observed. No facial paralysis and other lower cranial nerve dysfunction were observed. No postoperative complications such as cerebrospinal fluid leakage occurred. Conclusions: Using an angled endoscope combined with microscope in microvascular decompression in hemifacial spasmis is safe and effective.

Strategy for facial nerve management during surgical removal of benign jugular foramen tumors: Outcomes and indications.

OBJECTIVE: Classical surgical management of jugular foramen (JF) tumors usually requires facial nerve rerouting which results in permanent facial palsy in most patients. The purpose of the article is to study the outcomes of different rerouting techniques, and to discuss their indications. MATERIAL AND METHODS: We retrospectively reviewed 98 patients with JF tumors operated at our center between January 2008 and December 2016 using different surgical approaches with the following procedures for facial nerve management: total anterior rerouting (TR), partial anterior rerouting (PR), and fallopian bridge (FB) technique. The data for facial nerve management, surgical outcome and postoperative facial nerve function were collected from the medical records. RESULTS: In the study, there were 48 males and 50 females. Of them, 61 (62.2%) were jugular paragangliomas, 22 (22.4%) schwannomas, and 15 (15.3%) meningiomas. Total tumor removal was achieved in 95 (96.9%) patients, while near-total removal was achieved in 3 (3.1%) paragangliomas. TR was applied in 31 (31.6%) patients with PR in 26 (26.5%) patients, and FB in 41 (41.8%) patients. The mean follow-up duration was 39.4±22.6 months, and 2 recurrences of paragangliomas were observed. Seventy-five patients (76.5%) had good facial function (HB I-II) at 1 year after surgery, the patients who received a TR approach presented significantly less HB I-II FN function (48.4%) than those with PR (82.6%, P<0.05) or those with FB technique (95.1%, P<0.001). 21 patients (21.4%) presented new-onset lower cranial nerve dysfunction of which 13 recovered at 1 year after surgery. CONCLUSION: Facial nerve management in JF tumors should be tailored individually. No-rerouting methods, such as the fallopian bridge technique, bring significantly better results in terms of facial nerve function, which might be performed first during surgery; its indication is based mainly on the tumor type and extent.

Advancing osteochondral tissue engineering: bone morphogenetic protein, transforming growth factor, and fibroblast growth factor signaling drive ordered differentiation of periosteal cells resulting in stable cartilage and bone formation in vivo.

BACKGROUND: Chondrogenic mesenchymal stem cells (MSCs) have not yet been used to address the clinical demands of large osteochondral joint surface defects. In this study, self-assembling tissue intermediates (TIs) derived from human periosteum-derived stem/progenitor cells (hPDCs) were generated and validated for stable cartilage formation in vivo using two different animal models. METHODS: hPDCs were aggregated and cultured in the presence of a novel growth factor (GF) cocktail comprising of transforming growth factor (TGF)-ß1, bone morphogenetic protein (BMP)2, growth differentiation factor (GDF)5, BMP6, and fibroblast growth factor (FGF)2. Quantitative polymerase chain reaction (PCR) and immunohistochemistry were used to study in vitro differentiation. Aggregates were then implanted ectopically in nude mice and orthotopically in critical-size osteochondral defects in nude rats and evaluated by microcomputed tomography (µCT) and immunohistochemistry. RESULTS: Gene expression analysis after 28 days of in vitro culture revealed the expression of early and late chondrogenic markers and a significant upregulation of NOGGIN as compared to human articular chondrocytes (hACs). Histological examination revealed a bilayered structure comprising of chondrocytes at different stages of maturity. Ectopically, TIs generated both bone and mineralized cartilage at 8 weeks after implantation. Osteochondral defects treated with TIs displayed glycosaminoglycan (GAG) production, type-II collagen, and lubricin expression. Immunostaining for human nuclei protein suggested that hPDCs contributed to both subchondral bone and articular cartilage repair. CONCLUSION: Our data indicate that in vitro derived osteochondral-like tissues can be generated from hPDCs, which are capable of producing bone and cartilage ectopically and behave orthotopically as osteochondral units.

Simultaneous supervision by microscope of endoscope-assisted microsurgery via presigmoid retrolabyrinthine approach: A pilot study.

OBJECTIVE: To examine the impact of integrated hybrid operating rooms for endoscope-assisted microsurgery using the presigmoid retrolabyrinthine (RL) approach, and to determine the value of simultaneous supervision of skull base endoscopic procedures by microscope. MATERIAL AND METHODS: We retrospectively reviewed endoscope-assisted surgery using the RL approach at our institution between September 2013 and January 2017. The simultaneous supervision of endoscopic procedures by microscope was realized using the integrated hybrid system. Intra- or postoperative complications and surgical outcomes were analyzed. All patients were followed for at least 1 year. RESULTS: In total, 32 patients were studied: 4 vestibular schwannomas, 5 cholesteatomas, 8 hemifacial spasms, 5 glossopharyngeal neuralgias, and 10 Ménière's disease. In patients with vestibular schwannoma or cholesteatoma, complete removal was performed in all patients. In patients with Ménière's disease, hemifacial spasm or glossopharyngeal neuralgia, satisfactory symptom relief was achieved in all patients. Two (6.3%) patients had hearing loss after surgery which did not recover. One (3.1%) patient with vestibular schwannoma had mild facial palsy (HB III) at 2 weeks after the operation and recovered to near normal facial nerve function (HB II) at 1 year after surgery. No permanent or transient dysfunction of the trigeminal nerve or the lower cranial nerves was observed during follow-up. No complications such as cerebrospinal fluid (CSF) leakage or meningitis were observed. CONCLUSION: The endoscope provided a clearer and larger view, which solved the limitations of surgery using the RL approach. Endoscopic surgery under simultaneous supervision by microscope was safe and efficient in hearing preservation as well as in preservation of facial nerve function. An integrated operation room provided better support and the ability to switch quickly between these various complex devices.

Effect of alpha-tocopherol on restenosis after angioplasty in a model of experimental atherosclerosis.

The ability of alpha-tocopherol to reduce restenosis after angioplasty was tested in a rabbit model in which angioplasty was performed on established atherosclerotic lesions. Lesions induced by 4 wk of cholesterol feeding after focal desiccation of femoral arteries were balloon dilated. 3 wk after angioplasty, angiographically determined minimum luminal diameters were less in the untreated group (0.80 +/- 0.51 mm) than in the group treated with oral alpha-tocopherol beginning 19 d before angioplasty (1.38 +/- 0.29 mm; P < 0.01). The cross-sectional area of the intima-media was greater in the untreated group (1.18 +/- 0.48 mm2) than in the alpha-tocopherol group (0.62 +/- 0.25 mm2, P < 0.0001). These differences were not due to vasoconstriction or altered plasma cholesterol. Alpha-tocopherol thus reduced restenosis after angioplasty in this model. In rabbit vascular smooth muscle cells, oxidized low density lipoprotein stimulated DNA synthesis. Alpha-tocopherol treatment inhibited DNA synthesis stimulated by oxidized low density lipoprotein, but not by serum. The findings are consistent with the hypothesis that oxidized lipids can stimulate hyperplasia and that antioxidants may limit hyperplasia by inhibiting either the oxidation or the proliferative effects of oxidants on cells.

Combining microCT-based characterization with empirical modelling as a robust screening approach for the design of optimized CaP-containing scaffolds for progenitor cell-mediated bone formation.

Biomaterials are a key ingredient to the success of bone tissue engineering (TE), which focuses on the healing of bone defects by combining scaffolds with cells and/or growth factors. Due to the widely variable material characteristics and patient-specificities, however, current bone TE strategies still suffer from low repeatability and lack of robustness, which hamper clinical translation. Hence, optimal TE construct (i.e. cells and scaffold) characteristics are still under debate. This study aimed to reduce the material-specific variability for cell-based construct design, avoiding trial-and-error, by combining microCT characterization and empirical modelling as an innovative and robust screening approach. Via microCT characterization we have built a quantitative construct library of morphological and compositional properties of six CE approved CaP-based scaffolds (CopiOs®, BioOss™, Integra Mozaik™, chronOS Vivify, MBCP™ and ReproBone™), and of their bone forming capacity and in vivo scaffold degradation when combined with human periosteal derived cells (hPDCs). The empirical model, based on the construct library, allowed identification of the construct characteristics driving optimized bone formation, i.e. (a) the percentage of ß-TCP and dibasic calcium phosphate, (b) the concavity of the CaP structure, (c) the average CaP structure thickness and (d) the seeded cell amount (taking into account the seeding efficiency). Additionally, the model allowed to quantitatively predict the bone forming response of different hPDC-CaP scaffold combinations, thus providing input for a more robust design of optimized constructs and avoiding trial-and error. This could improve and facilitate clinical translation. STATEMENT OF SIGNIFICANCE: Biomaterials that support regenerative processes are a key ingredient for successful bone tissue engineering (TE). However, the optimal scaffold structure is still under debate. In this study, we have provided a useful innovative approach for robust screening of potential biomaterials or constructs (i.e. scaffolds seeded with cells and/or growth factors) by combining microCT characterization with empirical modelling. This novel approach leads to a better insight in the scaffold parameters influencing progenitor cell-mediated bone formation. Additionally, it serves as input for more controlled and robust design of optimized CaP-containing bone TE scaffolds. Hence, this novel approach could improve and facilitate clinical translation.

The late increase in intracellular free radical oxygen species during apoptosis is associated with cytochrome c release, caspase activation, and mitochondrial dysfunction.

Mitochondria play central roles in cellular metabolism and apoptosis and are a major source of reactive oxygen species (ROS). We investigated the role of ROS and mitochondria in radiation-induced apoptosis in multiple myeloma cells. Two distinct levels of ROS were generated following irradiation: a small increase observed early, and a pronounced late increase, associated with depletion of reduced glutathione (GSH) and collapse of mitochondrial membrane potential (deltapsi(m)). Exogenous ROS and caspase-3 induced deltapsi(m) drop and cytochrome c release from mitochondria, which could be prevented by molecular (dominant-negative caspase-9) and pharmacologic (zVAD-fmk) caspase inhibitors and overexpression of Bcl-2. Exogenous ROS also induced mitochondrial permeability transition (PT) pore opening and cytochrome c release in isolated mitochondria, which could be blocked by inhibition of PT with cyclosporin A. These results indicate that the late ROS production is associated with increased PT pore opening and decreased deltapsi(m), and GSH, events associated with caspase activation and cytochrome c release.

Cell based advanced therapeutic medicinal products for bone repair: Keep it simple?

The development of cell based advanced therapeutic medicinal products (ATMPs) for bone repair has been expected to revolutionize the health care system for the clinical treatment of bone defects. Despite this great promise, the clinical outcomes of the few cell based ATMPs that have been translated into clinical treatments have been far from impressive. In part, the clinical outcomes have been hampered because of the simplicity of the first wave of products. In response the field has set-out and amassed a plethora of complexities to alleviate the simplicity induced limitations. Many of these potential second wave products have remained "stuck" in the development pipeline. This is due to a number of reasons including the lack of a regulatory framework that has been evolving in the last years and the shortage of enabling technologies for industrial manufacturing to deal with these novel complexities. In this review, we reflect on the current ATMPs and give special attention to novel approaches that are able to provide complexity to ATMPs in a straightforward manner. Moreover, we discuss the potential tools able to produce or predict 'goldilocks' ATMPs, which are neither too simple nor too complex.

Effects of anodizing parameters and heat treatment on nanotopographical features, bioactivity, and cell culture response of additively manufactured porous titanium.

Anodizing could be used for bio-functionalization of the surfaces of titanium alloys. In this study, we use anodizing for creating nanotubes on the surface of porous titanium alloy bone substitutes manufactured using selective laser melting. Different sets of anodizing parameters (voltage: 10 or 20V anodizing time: 30min to 3h) are used for anodizing porous titanium structures that were later heat treated at 500°C. The nanotopographical features are examined using electron microscopy while the bioactivity of anodized surfaces is measured using immersion tests in the simulated body fluid (SBF). Moreover, the effects of anodizing and heat treatment on the performance of one representative anodized porous titanium structures are evaluated using in vitro cell culture assays using human periosteum-derived cells (hPDCs). It has been shown that while anodizing with different anodizing parameters results in very different nanotopographical features, i.e. nanotubes in the range of 20 to 55nm, anodized surfaces have limited apatite-forming ability regardless of the applied anodizing parameters. The results of in vitro cell culture show that both anodizing, and thus generation of regular nanotopographical feature, and heat treatment improve the cell culture response of porous titanium. In particular, cell proliferation measured using metabolic activity and DNA content was improved for anodized and heat treated as well as for anodized but not heat-treated specimens. Heat treatment additionally improved the cell attachment of porous titanium surfaces and upregulated expression of osteogenic markers. Anodized but not heat-treated specimens showed some limited signs of upregulated expression of osteogenic markers. In conclusion, while varying the anodizing parameters creates different nanotube structure, it does not improve apatite-forming ability of porous titanium. However, both anodizing and heat treatment at 500°C improve the cell culture response of porous titanium.

Germline mutations and genotype-phenotype associations in head and neck paraganglioma patients with negative family history in China.

The aim of this study was to assess the frequency of germline mutations and to explore genotype-phenotype associations in Chinese head and neck paraganglioma (HNPGL) patients without family history. Twenty-six Chinese patients with a diagnosis of HNPGL（14 male and 12 female, respectively）were recruited, who were followed up from 2000 to 2012. Genomic DNA was obtained from resected tumor tissues and peripheral blood samples. Seven genes, Succinate dehydrogenase complex A,B,C,D (SDHA, SDHB, SDHC, SDHD), succinate dehydrogenase complex assembly factor 2 (SDHAF2), TMEM127 (transmembrane protein 127) and VHL (Von Hippel-Lindau), were screened by direct sequencing and multiplex ligation-dependent probe amplification (MLPA) was performed to search for potential large deletions or duplications of SDHB, SDHC, SDHD, SDHAF1 and SDHAF2. The total frequency of germline mutations was 30.8% (8/26), including 5 cases with missense mutation p.Met1Ile in SDHD, 1 case with missense mutation p.Tyr216Cys in SDHB, and 1 case with a novel truncation mutation p.Gln44Ter in SDHAF2. MLPA showed one patient with malignant HNPGL had heterozygous deletions of exon1, 2, 3, 7 and 8 in SDHB. Mutations in SDHD were the leading cause of HNPGL in this study. Mutation carriers were younger than non-mutation carriers (p < 0.01) and more likely to suffer from multiple tumors (p = 0.048), especially with mutations in SDHD. The presence of mutation was associated with the development of larger tumors (p = 0.021). This study confirmed that the missense mutation p.Met1Ile at the start codon in SDHD was a hotspot in chinese patients with HNPGLs. We recommend genetic analysis in patients below 45 years, especially SDHD gene.

Gelatin functionalised porous titanium alloy implants for orthopaedic applications.

In the present work, we studied the immobilisation of the biopolymer gelatin onto the surface of three dimensional (3D) regular Ti6Al4V porous implants to improve their surface bio-activity. The successful immobilisation of the gelatin coating was made possible by a polydopamine interlayer, a polymer coating inspired by the adhesive nature of mussels. The presence of both coatings was first optimised on two dimensional titanium (2D Ti) substrates and confirmed by different techniques including X-ray photelectron spectroscopy, contact angle measurements, atomic force microscopy and fluorescence microscopy. Results showed homogeneous coatings that are stable for at least 24h in phosphate buffer at 37°C. In a next step, the coating procedure was successfully transferred to 3D Ti6Al4V porous implants, which indicates the versatility of the applied coating procedure with regard to complex surface morphologies. Furthermore, the bio-activity of these stable gelatin coatings was enhanced by applying a third and final coating using the cell-attractive protein fibronectin. The reproducible immobilisation process allowed for a controlled biomolecule presentation to the surrounding tissue. This newly developed coating procedure outperformed the previously reported silanisation procedure for immobilising gelatin. In vitro cell adhesion and culture studies with human periosteum-derived cells showed that the investigated coatings did not compromise the biocompatible nature of Ti6Al4V porous implants, but no distinct biological differences between the coatings were found.

A computational model for cell/ECM growth on 3D surfaces using the level set method: a bone tissue engineering case study.

Three-dimensional open porous scaffolds are commonly used in tissue engineering (TE) applications to provide an initial template for cell attachment and subsequent cell growth and construct development. The macroscopic geometry of the scaffold is key in determining the kinetics of cell growth and thus in vitro 'tissue' formation. In this study, we developed a computational framework based on the level set methodology to predict curvature-dependent growth of the cell/extracellular matrix domain within TE constructs. Scaffolds with various geometries (hexagonal, square, triangular) and pore sizes (500 and 1,000 [Formula: see text]m) were produced in-house by additive manufacturing, seeded with human periosteum-derived cells and cultured under static conditions for 14 days. Using the projected tissue area as an output measure, the comparison between the experimental and the numerical results demonstrated a good qualitative and quantitative behavior of the framework. The model in its current form is able to provide important spatio-temporal information on final shape and speed of pore-filling of tissue-engineered constructs by cells and extracellular matrix during static culture.

The effect of pore geometry on the in vitro biological behavior of human periosteum-derived cells seeded on selective laser-melted Ti6Al4V bone scaffolds.

The specific aim of this study was to gain insight into the influence of scaffold pore size, pore shape and permeability on the in vitro proliferation and differentiation of three-dimensional (3-D) human periosteum-derived cell (hPDC) cultures. Selective laser melting (SLM) was used to produce six distinct designed geometries of Ti6Al4V scaffolds in three different pore shapes (triangular, hexagonal and rectangular) and two different pore sizes (500 µm and 1000 µm). All scaffolds were characterized by means of two-dimensional optical microscopy, 3-D microfocus X-ray computed tomography (micro-CT) image analysis, mechanical compression testing and computational fluid dynamical analysis. The results showed that SLM was capable of producing Ti6Al4V scaffolds with a broad range of morphological and mechanical properties. The in vitro study showed that scaffolds with a lower permeability gave rise to a significantly higher number of cells attached to the scaffolds after seeding. Qualitative analysis by means of live/dead staining and scanning electron micrography showed a circular cell growth pattern which was independent of the pore size and shape. This resulted in pore occlusion which was found to be the highest on scaffolds with 500 µm hexagonal pores. Interestingly, pore size but not pore shape was found to significantly influence the growth of hPDC on the scaffolds, whereas the differentiation of hPDC was dependent on both pore shape and pore size. The results showed that, for SLM-produced Ti6Al4V scaffolds with specific morphological and mechanical properties, a functional graded scaffold will contribute to enhanced cell seeding and at the same time can maintain nutrient transport throughout the whole scaffold during in vitro culturing by avoiding pore occlusion.

Current views on calcium phosphate osteogenicity and the translation into effective bone regeneration strategies.

Calcium phosphate (CaP) has traditionally been used for the repair of bone defects because of its strong resemblance to the inorganic phase of bone matrix. Nowadays, a variety of natural or synthetic CaP-based biomaterials are produced and have been extensively used for dental and orthopaedic applications. This is justified by their biocompatibility, osteoconductivity and osteoinductivity (i.e. the intrinsic material property that initiates de novo bone formation), which are attributed to the chemical composition, surface topography, macro/microporosity and the dissolution kinetics. However, the exact molecular mechanism of action is unknown. This review paper first summarizes the most important aspects of bone biology in relation to CaP and the mechanisms of bone matrix mineralization. This is followed by the research findings on the effects of calcium (Ca²âº) and phosphate (PO4³â») ions on the migration, proliferation and differentiation of osteoblasts during in vivo bone formation and in vitro culture conditions. Further, the rationale of using CaP for bone regeneration is explained, focusing thereby specifically on the material's osteoinductive properties. Examples of different material forms and production techniques are given, with the emphasis on the state-of-the art in fine-tuning the physicochemical properties of CaP-based biomaterials for improved bone induction and the use of CaP as a delivery system for bone morphogenetic proteins. The use of computational models to simulate the CaP-driven osteogenesis is introduced as part of a bone tissue engineering strategy in order to facilitate the understanding of cell-material interactions and to gain further insight into the design and optimization of CaP-based bone reparative units. Finally, limitations and possible solutions related to current experimental and computational techniques are discussed.

Designing optimal calcium phosphate scaffold-cell combinations using an integrative model-based approach.

Bone formation is a very complex physiological process, involving the participation of many different cell types and regulated by countless biochemical, physical and mechanical factors, including naturally occurring or synthetic biomaterials. For the latter, calcium phosphate (CaP)-based scaffolds have proven to stimulate bone formation, but at present still result in a wide range of in vivo outcomes, which is partly related to the suboptimal use and combination with osteogenic cells. To optimize CaP scaffold selection and make their use in combination with cells more clinically relevant, this study uses an integrative approach in which mathematical modeling is combined with experimental research. This paper describes the development and implementation of an experimentally informed bioregulatory model of the effect of calcium ions released from CaP-based biomaterials on the activity of osteogenic cells and mesenchymal stem cell driven ectopic bone formation. The amount of bone formation predicted by the mathematical model corresponds to the amount measured experimentally under similar conditions. Moreover, the model is also able to qualitatively predict the experimentally observed impaired bone formation under conditions such as insufficient cell seeding and scaffold decalcification. A strategy was designed in silico to overcome the negative influence of a low initial cell density on the bone formation process. Finally, the model was applied to design optimal combinations of calcium-based biomaterials and cell culture conditions with the aim of maximizing the amount of bone formation. This work illustrates the potential of mathematical models as research tools to design more efficient and cell-customized CaP scaffolds for bone tissue engineering applications.