Bayesian cell therapy process optimization.

Optimizing complex bioprocesses poses a significant challenge in several fields, particularly in cell therapy manufacturing. The development of customized, closed, and automated processes is crucial for their industrial translation and for addressing large patient populations at a sustainable price. Limited understanding of the underlying biological mechanisms, coupled with highly resource-intensive experimentation, are two contributing factors that make the development of these next-generation processes challenging. Bayesian optimization (BO) is an iterative experimental design methodology that addresses these challenges, but has not been extensively tested in situations that require parallel experimentation with significant experimental variability. In this study, we present an evaluation of noisy, parallel BO for increasing noise levels and parallel batch sizes on two in silico bioprocesses, and compare it to the industry state-of-the-art. As an in vitro showcase, we apply the method to the optimization of a monocyte purification unit operation. The in silico results show that BO significantly outperforms the state-of-the-art, requiring approximately 50% fewer experiments on average. This study highlights the potential of noisy, parallel BO as valuable tool for cell therapy process development and optimization.

Large-scale progenitor cell expansion for multiple donors in a monitored hollow fibre bioreactor.

BACKGROUND AIMS: With the increasing scale in stem cell production, a robust and controlled cell expansion process becomes essential for the clinical application of cell-based therapies. The objective of this work was the assessment of a hollow fiber bioreactor (Quantum Cell Expansion System from Terumo BCT) as a cell production unit for the clinical-scale production of human periosteum derived stem cells (hPDCs). METHODS: We aimed to demonstrate comparability of bioreactor production to standard culture flask production based on a product characterization in line with the International Society of Cell Therapy in vitro benchmarks and supplemented with a compelling quantitative in vivo bone-forming potency assay. Multiple process read-outs were implemented to track process performance and deal with donor-to-donor-related variation in nutrient needs and harvest timing. RESULTS: The data show that the hollow fiber bioreactor is capable of robustly expanding autologous hPDCs on a clinical scale (yield between 316 million and 444 million cells starting from 20 million after ± 8 days of culture) while maintaining their in vitro quality attributes compared with the standard flask-based culture. The in vivo bone-forming assay on average resulted in 10.3 ± 3.7% and 11.0 ± 3.8% newly formed bone for the bioreactor and standard culture flask respectively. The analysis showed that the Quantum system provides a reproducible cell expansion process in terms of yields and culture conditions for multiple donors.

Expansion of murine periosteal progenitor cells with fibroblast growth factor 2 reveals an intrinsic endochondral ossification program mediated by bone morphogenetic protein 2.

The preservation of the bone-forming potential of skeletal progenitor cells during their ex vivo expansion remains one of the major challenges for cell-based bone regeneration strategies. We report that expansion of murine periosteal cells in the presence of FGF2, a signal present during the early stages of fracture healing, is necessary and sufficient to maintain their ability to organize in vivo into a cartilage template which gives rise to mature bone. Implantation of FGF2-primed cells in a large bone defect in mice resulted in complete healing, demonstrating the feasibility of using this approach for bone tissue engineering purposes. Mechanistically, the enhanced endochondral ossification potential of FGF2-expanded periosteal cells is predominantly driven by an increased production of BMP2 and is additionally linked to an improved preservation of skeletal progenitor cells in the cultures. This characteristic is unique for periosteal cells, as FGF2-primed bone marrow stromal cells formed significantly less bone and progressed exclusively through the intramembranous pathway, revealing essential differences between both cell pools. Taken together, our findings provide insight in the molecular regulation of fracture repair by identifying a unique interaction between periosteal cells and FGF2. These insights may promote the development of cell-based therapeutic strategies for bone regeneration which are independent of the in vivo use of growth factors, thus limiting undesired side effects.

Spatial optimization in perfusion bioreactors improves bone tissue-engineered construct quality attributes.

Perfusion bioreactors have shown great promise for tissue engineering applications providing a homogeneous and consistent distribution of nutrients and flow-induced shear stresses throughout tissue-engineered constructs. However, non-uniform fluid-flow profiles found in the perfusion chamber entrance region have been shown to affect tissue-engineered construct quality characteristics during culture. In this study a whole perfusion and construct, three dimensional (3D) computational fluid dynamics approach was used in order to optimize a critical design parameter such as the location of the regular pore scaffolds within the perfusion bioreactor chamber. Computational studies were coupled to bioreactor experiments for a case-study flow rate. Two cases were compared in the first instance seeded scaffolds were positioned immediately after the perfusion chamber inlet while a second group was positioned at the computationally determined optimum distance were a steady state flow profile had been reached. Experimental data showed that scaffold location affected significantly cell content and neo-tissue distribution, as determined and quantified by contrast enhanced nanoCT, within the constructs both at 14 and 21 days of culture. However, gene expression level of osteopontin and osteocalcin was not affected by the scaffold location. This study demonstrates that the bioreactor chamber environment, incorporating a scaffold and its location within it, affects the flow patterns within the pores throughout the scaffold requiring therefore dedicated optimization that can lead to bone tissue engineered constructs with improved quality attributes.

Engineering vascularized bone: osteogenic and proangiogenic potential of murine periosteal cells.

One of the key challenges in bone tissue engineering is the timely formation of blood vessels that promote the survival of the implanted cells in the construct. Fracture healing largely depends on the presence of an intact periosteum but it is still unknown whether periosteum-derived cells (PDC) are critical for bone repair only by promoting bone formation or also by inducing neovascularization. We first established a protocol to specifically isolate murine PDC (mPDC) from long bones of adult mice. Mesenchymal stem cells were abundantly present in this cell population as more than 50% of the mPDC expressed mesenchymal markers (CD73, CD90, CD105, and stem cell antigen-1) and the cells exhibited trilineage differentiation potential (chondrogenic, osteogenic, and adipogenic). When transplanted on a collagen-calcium phosphate scaffold in vivo, mPDC attracted numerous blood vessels and formed mature bone which comprises a hematopoiesis-supportive stroma. We explored the proangiogenic properties of mPDC using in vitro culture systems and showed that mPDC promote the survival and proliferation of endothelial cells through the production of vascular endothelial growth factor. Coimplantation with endothelial cells demonstrated that mPDC can enhance vasculogenesis by adapting a pericyte-like phenotype, in addition to their ability to stimulate blood vessel ingrowth from the host. In conclusion, these findings demonstrate that periosteal cells contribute to fracture repair, not only through their strong osteogenic potential but also through their proangiogenic features and thus provide an ideal cell source for bone regeneration therapies.

Multiplexed protein detection using an affinity aptamer amplification assay.

Affinity probe capillary electrophoresis (APCE) is potentially one of the most versatile technologies for protein diagnostics, offering an excellent balance between robustness, analysis speed and sensitivity. Combining the immunosensing and separating strength of capillary electrophoresis with the signal enhancement power of nucleic acid amplification, aptamers can further push the analytical limits of APCE to offer ultrasensitive, multiplexed detection of protein biomarkers, even when differences in electrophoretic mobility between the different aptamer-target complexes are limited. It is demonstrated how, through careful selection of experimental parameters, simultaneous detection of picomolar levels of three target proteins can be achieved even with aptamers that were initially selected under very different conditions and further taking into account that the aptamers need to be modified to allow successful PCR amplification. Aptamer-enhanced APCE offers limits of detection that are orders of magnitude lower than those that can be achieved through traditional capillary electrophoresis-based immunosensing. With recent developments in aptamer selection that for the first time realise the promise of aptamers as easily accessible, high affinity recognition molecules, it can therefore be envisioned that aptamer-enhanced APCE on parallel microfluidic platforms can be the basis for a truly high-throughput multiplexed proteomics platform, rivalling genetic screening for the first time.

Mechanical properties and cytocompatibility of dense and porous Zn produced by laser powder bed fusion for biodegradable implant applications.

In this work, the macrotexture of dense Zn produced by laser powder bed fusion (LPBF) was studied and the mechanical properties for different tensile bar orientations were measured. The compressive strength of LPBF Zn scaffolds with five different unit cells was measured for a relative density of 20-51%. In addition, the response of mesenchymal stem cells to the LPBF Zn scaffolds was studied. The elastic modulus and yield strength of dense LPBF Zn were 110.0 ± 0.2 GPa and 78.0 ± 0.4 MPa, respectively in the vertical and 81.0 ± 0.4 GPa and 55.0 ± 0.7 MPa in the horizontal direction. This could be explained by the preferential orientation of the 〈0001〉 direction in the building plane. For LPBF Zn scaffolds, the plateau stress for the different unit cells varied between 8 and 33 MPa for a 30% relative density. Calcein staining, lactate production and DNA measurements over a 13-day period showed that mesenchymal stem cell viability was low for Zn scaffolds. This work forms a basis for further research into the LPBF texture formation of metals with hexagonal crystal structure, guides implant designers in scaffold unit cell and relative density selection and motivates further research into the cytocompatibility of LPBF Zn. STATEMENT OF SIGNIFICANCE: Laser powder bed fusion (LPBF) is a manufacturing technology which allows the seamless combination of porous and non-porous volumes in a metallic implant and is used in the orthopedic manufacturing industry today. The production of highly dense Zn with LPBF has been described earlier, but the mechanical properties of the resulting material have not been studied in detail yet. This study is the first to report on (i) the influence of different scanning strategies on the macrotexture of dense LPBF Zn and the resulting anisotropy of its mechanical properties, (ii) the relationship between the relative density and strength for LPBF Zn scaffolds with five different unit cells and (iii) the in vitro response of mesenchymal stem cells to these scaffolds.

Bone augmentation with autologous periosteal cells and two different calcium phosphate scaffolds under an occlusive titanium barrier: an experimental study in rabbits.

BACKGROUND: This study used a tissue-engineering approach, which combined autologous periosteal cells with a scaffold material, to promote bone augmentation under an occlusive titanium barrier that was placed on the skull of rabbits. Because the cell-matrix interaction is of key importance in tissue engineering, two different calcium phosphate-based scaffolds were seeded with autologous periosteal cells. One scaffold contained hydroxyapatite, tricalcium phosphate, and collagen; the other scaffold was a beta-tricalcium phosphate structure. METHODS: The experiment involved 38 rabbits divided into five groups: the two different scaffolds with and without cells and a blood clot only. Prior to implantation, autologous periosteal cells were harvested from the tibia by stripping the periosteum. Cells were cultured, and 1 day before the implantation approximately 20 million cells were collected and seeded onto the scaffolds. Two preformed dome-shaped full titanium barriers were placed subperiosteally onto the frontal and parietal bones of each rabbit. Before placement of the barriers, the different scaffolds, seeded with or without cells, were put on top of the skull. As a negative control, autologous blood was injected into the barriers. Histologic evaluation and histomorphometric analysis were performed after 12 weeks of undisturbed bone growth. Measurements involved the amounts of newly formed tissue and of new bone distinguishing between trabecular bone and osteoid. RESULTS: No significant differences were found between the four treatment groups (scaffolds with or without cells). However, the amount of new bone tissue found underneath the titanium barriers with scaffolds was significantly higher (P <0.04) than with a blood clot only. CONCLUSION: A better understanding of the mode of action is required to optimize tissue-engineering procedures before entering clinical applications.

* Harnessing the Osteogenicity of In Vitro Stem Cell-Derived Mineralized Extracellular Matrix as 3D Biotemplate to Guide Bone Regeneration.

Advanced biomaterials that are capable of guiding robust bone regeneration are highly demanded for translational therapy of bone defects or bone augmentation in clinics. One of the strategic approaches is to produce tissue engineering (TE) constructs that mediate bone regeneration by recapitulating the natural bone formation or healing process. In this study, we aimed at producing devitalized mineralized carriers with augmented bone forming capacity via a modified culture protocol (i.e., culture conditions with high calcium and/or phosphate concentrations) that first promotes cell growth and, subsequently, mineralized extracellular matrix (ECM) deposition by human periosteum-derived osteoprogenitor cells (hPDCs) on additive manufactured three-dimensional (3D) porous titanium (Ti)-based scaffolds. Qualitative and quantitative analysis was performed to characterize the physicochemical properties of the produced devitalized mineralized carriers, as well as their effects as carriers on in vitro cell growth and osteochondrogenic differentiation of hPDCs under a perfusion bioreactor culture set-up. The results showed that the modified culture protocol was useful to produce devitalized mineralized carriers with different amount, distribution, composition, and morphology of mineralized matrix that resembled hydroxyapatite, and exhibited different Ca2+ release kinetics, distinct human bone morphogenetic protein (hBMP)-2, human vascular endothelial growth factor (hVEGF) proteins, and collagen contents. The produced devitalized mineralized carriers supported 3D growth of hPDCs, with minor osteochondrogenic differentiation effects under the perfusion bioreactor culture condition. Subcutaneous implantation of hPDC-seeded devitalized mineralized carriers in athymic nude rats showed nearly five-fold augmentation in the ectopic bone-forming capacity, with no bone induction obtained for unseeded, devitalized mineralized carriers and plain Ti scaffolds. Implantation of devitalized mineralized carriers in critical-sized calvarial defects resulted in encouraging defect bridging as compared with limited defect bridging by plain Ti scaffolds or in empty defects. This defect bridging was not enhanced by implanting hPDC-seeded devitalized mineralized carriers. In conclusion, the investigated modified culture protocol was useful to produce devitalized mineralized carriers with augmented bone-forming capacity, which potentially could aid bone repair or augmentation in clinics.

Micro-CT-based screening of biomechanical and structural properties of bone tissue engineering scaffolds.

The development of successful scaffolds for bone tissue engineering requires a concurrent engineering approach that combines different research fields. In order to limit in vivo experiments and reduce trial and error research, a scaffold screening technique has been developed. In this protocol seven structural and three biomechanical properties of potential scaffold materials are quantified and compared to the desired values. The property assessment is done on computer models of the scaffolds, and these models are based on micro-CT images. As a proof of principle, three porous scaffolds were evaluated with this protocol: stainless steel, hydroxyapatite, and titanium. These examples demonstrate that the modelling technique is able to quantify important scaffold properties. Thus, a powerful technique for automated screening of bone tissue engineering scaffolds has been developed that in a later stage may be used to tailor the scaffold properties to specific requirements.

Early BMP, Wnt and Ca(2+)/PKC pathway activation predicts the bone forming capacity of periosteal cells in combination with calcium phosphates.

The development of osteoinductive calcium phosphate- (CaP) based biomaterials has, and continues to be, a major focus in the field of bone tissue engineering. However, limited insight into the spatiotemporal activation of signalling pathways has hampered the optimisation of in vivo bone formation and subsequent clinical translation. To gain further knowledge regarding the early molecular events governing bone tissue formation, we combined human periosteum derived progenitor cells with three types of clinically used CaP-scaffolds, to obtain constructs with a distinct range of bone forming capacity in vivo. Protein phosphorylation together with gene expression for key ligands and target genes were investigated 24 hours after cell seeding in vitro, and 3 and 12 days post ectopic implantation in nude mice. A computational modelling approach was used to deduce critical factors for bone formation 8 weeks post implantation. The combined Ca(2+)-mediated activation of BMP-, Wnt- and PKC signalling pathways 3 days post implantation were able to discriminate the bone forming from the non-bone forming constructs. Subsequently, a mathematical model able to predict in vivo bone formation with 96% accuracy was developed. This study illustrates the importance of defining and understanding CaP-activated signalling pathways that are required and sufficient for in vivo bone formation. Furthermore, we demonstrate the reliability of mathematical modelling as a tool to analyse and deduce key factors within an empirical data set and highlight its relevance to the translation of regenerative medicine strategies.

Large-Scale Mesenchymal Stem/Stromal Cell Expansion: A Visualization Tool for Bioprocess Comparison.

Large-scale and cost-effective cell expansion processes are a prerequisite for the clinical and commercial translation of cell-based therapies. A large variety of cell expansion processes are described in literature, utilizing different cell types, culture vessels, and medium formulations. Consequently there are no straightforward means for the comparison or benchmarking of these processes in terms of efficiency, scale, or costs. The purpose of this study was to systematically review the available mesenchymal stromal cell (MSC) expansion literature and develop an interactive visualization tool for comparing the expansion processes. By using this computational tool, process data could be concentrated, standardized, and analyzed to facilitate a more general understanding of the parameters that define a cell culture process, and in the future allow rational selection or design of these bioprocesses. Additionally, a set of bioprocess metrics were defined that assured the comparability between different processes. Currently, the literature-based data repository holds 73 individual cell expansion processes on seven different types of human MSCs in five different types of culture vessels. The visualization tool allowed benchmarking of these processes against each other, serving as a reference point for cell expansion process efficiency.

Quantitative Validation of the Presto Blue Metabolic Assay for Online Monitoring of Cell Proliferation in a 3D Perfusion Bioreactor System.

As the fields of tissue engineering and regenerative medicine mature toward clinical applications, the need for online monitoring both for quantitative and qualitative use becomes essential. Resazurin-based metabolic assays are frequently applied for determining cytotoxicity and have shown great potential for monitoring 3D bioreactor-facilitated cell culture. However, no quantitative correlation between the metabolic conversion rate of resazurin and cell number has been defined yet. In this work, we determined conversion rates of Presto Blue, a resazurin-based metabolic assay, for human periosteal cells during 2D and 3D static and 3D perfusion cultures. Our results showed that for the evaluated culture systems there is a quantitative correlation between the Presto Blue conversion rate and the cell number during the expansion phase with no influence of the perfusion-related parameters, that is, flow rate and shear stress. The correlation between the cell number and Presto Blue conversion subsequently enabled the definition of operating windows for optimal signal readouts. In conclusion, our data showed that the conversion of the resazurin-based Presto Blue metabolic assay can be used as a quantitative readout for online monitoring of cell proliferation in a 3D perfusion bioreactor system, although a system-specific validation is required.

Bioreactor-Based Online Recovery of Human Progenitor Cells with Uncompromised Regenerative Potential: A Bone Tissue Engineering Perspective.

The use of a 3D perfusion culture environment for stem cell expansion has been shown to be beneficial for maintenance of the original cell functionality but due to several system inherent characteristics such as the presence of extracellular matrix, the continued development and implementation of 3D perfusion bioreactor technologies is hampered. Therefore, this study developed a methodology for harvesting a progenitor cell population from a 3D open porous culture surface after expansion in a perfusion bioreactor and performed a functional characterization of the expanded cells. An initial screening showed collagenase to be the most interesting reagent to release the cells from the 3D culture surface as it resulted in high yields without compromising cell viability. Subsequently a Design of Experiment approach was used to obtain optimized 3D harvest conditions by assessing the interplay of flow rate, collagenase concentration and incubation time on the harvest efficiency, viability and single cell fraction. Cells that were recovered with the optimized harvest protocol, by perfusing a 880 U/ml collagenase solution for 7 hours at a flow rate of 4 ml/min, were thereafter functionally analyzed for their characteristics as expanded progenitor cell population. As both the in vitro tri-lineage differentiation capacity and the in vivo bone forming potential were maintained after 3D perfusion bioreactor expansion we concluded that the developed seeding, culture and harvest processes did not significantly compromise the viability and potency of the cells and can contribute to the future development of integrated bioprocesses for stem cell expansion.

Additively Manufactured Open-Cell Porous Biomaterials Made from Six Different Space-Filling Unit Cells: The Mechanical and Morphological Properties.

It is known that the mechanical properties of bone-mimicking porous biomaterials are a function of the morphological properties of the porous structure, including the configuration and size of the repeating unit cell from which they are made. However, the literature on this topic is limited, primarily because of the challenge in fabricating porous biomaterials with arbitrarily complex morphological designs. In the present work, we studied the relationship between relative density (RD) of porous Ti6Al4V EFI alloy and five compressive properties of the material, namely elastic gradient or modulus (Es20-70), first maximum stress, plateau stress, yield stress, and energy absorption. Porous structures with different RD and six different unit cell configurations (cubic (C), diamond (D), truncated cube (TC), truncated cuboctahedron (TCO), rhombic dodecahedron (RD), and rhombicuboctahedron (RCO)) were fabricated using selective laser melting. Each of the compressive properties increased with increase in RD, the relationship being of a power law type. Clear trends were seen in the influence of unit cell configuration and porosity on each of the compressive properties. For example, in terms of Es20-70, the structures may be divided into two groups: those that are stiff (comprising those made using C, TC, TCO, and RCO unit cell) and those that are compliant (comprising those made using D and RD unit cell).

Additively manufactured porous tantalum implants.

The medical device industry's interest in open porous, metallic biomaterials has increased in response to additive manufacturing techniques enabling the production of complex shapes that cannot be produced with conventional techniques. Tantalum is an important metal for medical devices because of its good biocompatibility. In this study selective laser melting technology was used for the first time to manufacture highly porous pure tantalum implants with fully interconnected open pores. The architecture of the porous structure in combination with the material properties of tantalum result in mechanical properties close to those of human bone and allow for bone ingrowth. The bone regeneration performance of the porous tantalum was evaluated in vivo using an orthotopic load-bearing bone defect model in the rat femur. After 12 weeks, substantial bone ingrowth, good quality of the regenerated bone and a strong, functional implant-bone interface connection were observed. Compared to identical porous Ti-6Al-4V structures, laser-melted tantalum shows excellent osteoconductive properties, has a higher normalized fatigue strength and allows for more plastic deformation due to its high ductility. It is therefore concluded that this is a first step towards a new generation of open porous tantalum implants manufactured using selective laser melting.

Multifactorial Optimization of Contrast-Enhanced Nanofocus Computed Tomography for Quantitative Analysis of Neo-Tissue Formation in Tissue Engineering Constructs.

To progress the fields of tissue engineering (TE) and regenerative medicine, development of quantitative methods for non-invasive three dimensional characterization of engineered constructs (i.e. cells/tissue combined with scaffolds) becomes essential. In this study, we have defined the most optimal staining conditions for contrast-enhanced nanofocus computed tomography for three dimensional visualization and quantitative analysis of in vitro engineered neo-tissue (i.e. extracellular matrix containing cells) in perfusion bioreactor-developed Ti6Al4V constructs. A fractional factorial 'design of experiments' approach was used to elucidate the influence of the staining time and concentration of two contrast agents (Hexabrix and phosphotungstic acid) and the neo-tissue volume on the image contrast and dataset quality. Additionally, the neo-tissue shrinkage that was induced by phosphotungstic acid staining was quantified to determine the operating window within which this contrast agent can be accurately applied. For Hexabrix the staining concentration was the main parameter influencing image contrast and dataset quality. Using phosphotungstic acid the staining concentration had a significant influence on the image contrast while both staining concentration and neo-tissue volume had an influence on the dataset quality. The use of high concentrations of phosphotungstic acid did however introduce significant shrinkage of the neo-tissue indicating that, despite sub-optimal image contrast, low concentrations of this staining agent should be used to enable quantitative analysis. To conclude, design of experiments allowed us to define the most optimal staining conditions for contrast-enhanced nanofocus computed tomography to be used as a routine screening tool of neo-tissue formation in Ti6Al4V constructs, transforming it into a robust three dimensional quality control methodology.

Revival of pure titanium for dynamically loaded porous implants using additive manufacturing.

Additive manufacturing techniques are getting more and more established as reliable methods for producing porous metal implants thanks to the almost full geometrical and mechanical control of the designed porous biomaterial. Today, Ti6Al4V ELI is still the most widely used material for porous implants, and none or little interest goes to pure titanium for use in orthopedic or load-bearing implants. Given the special mechanical behavior of cellular structures and the material properties inherent to the additive manufacturing of metals, the aim of this study is to investigate the properties of selective laser melted pure unalloyed titanium porous structures. Therefore, the static and dynamic compressive properties of pure titanium structures are determined and compared to previously reported results for identical structures made from Ti6Al4V ELI and tantalum. The results show that porous Ti6Al4V ELI still remains the strongest material for statically loaded applications, whereas pure titanium has a mechanical behavior similar to tantalum and is the material of choice for cyclically loaded porous implants. These findings are considered to be important for future implant developments since it announces a potential revival of the use of pure titanium for additively manufactured porous implants.

Osteostatin-coated porous titanium can improve early bone regeneration of cortical bone defects in rats.

A promising bone graft substitute is porous titanium. Porous titanium, produced by selective laser melting (SLM), can be made as a completely open porous and load-bearing scaffold that facilitates bone regeneration through osteoconduction. In this study, the bone regenerative capacity of porous titanium is improved with a coating of osteostatin, an osteoinductive peptide that consists of the 107-111 domain of the parathyroid hormone (PTH)-related protein (PTHrP), and the effects of this osteostatin coating on bone regeneration were evaluated in vitro and in vivo. SLM-produced porous titanium received an alkali-acid-heat treatment and was coated with osteostatin through soaking in a 100 nM solution for 24 h or left uncoated. Osteostatin-coated scaffolds contained ∼0.1 µg peptide/g titanium, and in vitro 81% was released within 24 h. Human periosteum-derived osteoprogenitor cells cultured on osteostatin-coated scaffolds did not induce significant changes in osteogenic (alkaline phosphatase [ALP], collagen type 1 [Col1], osteocalcin [OCN], runt-related transcription factor 2 [Runx2]), or angiogenic (vascular endothelial growth factor [VEGF]) gene expression; however, it resulted in an upregulation of osteoprotegerin (OPG) gene expression after 24 h and a lower receptor activator of nuclear factor kappa-B ligand (RankL):OPG mRNA ratio. In vivo, osteostatin-coated, porous titanium implants increased bone regeneration in critical-sized cortical bone defects (p=0.005). Bone regeneration proceeded until 12 weeks, and femurs grafted with osteostatin-coated implants and uncoated implants recovered, respectively, 66% and 53% of the original femur torque strength (97±31 and 77±53 N·mm, not significant). In conclusion, the osteostatin coating improved bone regeneration of porous titanium. This effect was initiated after a short burst release and might be related to the observed in vitro upregulation of OPG gene expression by osteostatin in osteoprogenitor cells. Long-term beneficial effects of osteostatin-coated, porous titanium implants on bone regeneration or mechanical strength were not established here and may require optimization of the pace and dose of osteostatin release.

Endothelial Msx1 transduces hemodynamic changes into an arteriogenic remodeling response.

Collateral remodeling is critical for blood flow restoration in peripheral arterial disease and is triggered by increasing fluid shear stress in preexisting collateral arteries. So far, no arterial-specific mediators of this mechanotransduction response have been identified. We show that muscle segment homeobox 1 (MSX1) acts exclusively in collateral arterial endothelium to transduce the extrinsic shear stimulus into an arteriogenic remodeling response. MSX1 was specifically up-regulated in remodeling collateral arteries. MSX1 induction in collateral endothelial cells (ECs) was shear stress driven and downstream of canonical bone morphogenetic protein-SMAD signaling. Flow recovery and collateral remodeling were significantly blunted in EC-specific Msx1/2 knockout mice. Mechanistically, MSX1 linked the arterial shear stimulus to arteriogenic remodeling by activating the endothelial but not medial layer to a proinflammatory state because EC but not smooth muscle cellMsx1/2 knockout mice had reduced leukocyte recruitment to remodeling collateral arteries. This reduced leukocyte infiltration in EC Msx1/2 knockout mice originated from decreased levels of intercellular adhesion molecule 1 (ICAM1)/vascular cell adhesion molecule 1 (VCAM1), whose expression was also in vitro driven by promoter binding of MSX1.