Engineered 3D Silk‐Based Metastasis Models: Interactions Between Human Breast Adenocarcinoma,Mesenchymal Stem Cells and Osteoblast‐Like Cells

Bone metastasis occurs in 70% of breast cancer patients and is a frequent cause of morbidity in cancer patients. A delicate balance exists in the bone microenvironment, but the functional dynamics underlying the tumor cell‐microenvironment interactions remain poorly understood. 3D in vitro model systems of metastasis can throw new light on this phenomenon. Silk protein fibroin scaffolds, are cytocompatible for 3D cancer cell culture. They are structurally more resistant to protease degradation than other native biomaterials making these matrices suitable for cancer modeling. In this report, human breast adenocarcinoma cells, human osteoblast like cells and mesenchymal stem cells are co‐cultered. Cancer cells and osteoblast‐like cells are found to interact through secreted products. Decreased population of osteoblast‐like cells and mineralization of extracellular matrix are observed as a result of co‐culture. Significantly increased migration of breast cancer cells is observed in the bone‐like constructs than in non‐seeded scaffolds. The co‐culture constructs show significant increase in drug resistance, invasiveness and angiogenicity. Co‐culture of breast cancer cells with osteoblast like cells and mesenchymal stem cells also indicate that the interaction of cancer cells with bone microenvironment varies with spatial organization, presence of osteogenic factors as well as stromal cell type. Here, results show that 3D in vitro co‐culture models is possibly a better system to study and target cancer progression.     

Injectable Peptide Decorated Functional Nanofibrous Hollow Microspheres to Direct Stem Cell Differentiation and Tissue Regeneration

Injectable microspheres are attractive stem cell carriers for minimally invasive procedures. For tissue regeneration, the microspheres need to present the critical cues to properly direct stem cell differentiation. In natural extracellular matrix (ECM), growth factors (GFs) and collagen nanofibers provide critical chemical and physical cues. However, there have been no reported technologies that integrate synthetic nanofibers and GFs into injectable microspheres. In this study, functional nanofibrous hollow microspheres (FNF‐HMS), which can covalently bind GF‐mimicking peptides, are synthesized. Two different GF‐mimicking peptides, Transforming Growth Factor‐β1 mimicking peptide Cytomodulin (CM) and Bone Morphogenetic Protein‐2 mimicking peptide P24, are separately conjugated onto the FNF‐HMS to induce distinct differentiation pathways of rabbit bone marrow‐derived mesenchymal stem cells (BMSCs). While no existing biomaterials are reported to successfully deliver CM to induce chondrogenesis, the developed FNF‐HMS are shown to effectively present CM to BMSCs and successfully induced their chondrogenesis for ­cartilage formation in both in vitro and in vivo studies. In addition, P24 is conjugated onto the newly developed FNF‐HMS and is capable of retaining its bioactivity and inducing ectopic bone formation in nude mice. These results demonstrate that the novel FNF‐HMS can effectively deliver GF‐mimicking peptides to modulate stem cell fate and tissue regeneration.     

A Novel Hydrogel Surface Grafted With Dual Functional Peptides for Sustaining Long‐Term Self‐Renewal of Human Induced Pluripotent Stem Cells and Manipulating Their Osteoblastic Maturation

Realizing the clinical potential of human induced pluripotent stem cells (hiPSCs) in bone regenerative medicine requires the development of safe and chemically defined biomaterials for expansion of hiPSCs followed by directing their lineage commitment to osteoblasts. In this study, novel multipurpose peptide‐presenting hydrogel surfaces are prepared on common tissue culture plates via carboxymethyl chitosan grafting and subsequent immobilization of two functional peptides allowing for in vitro feeder‐free culture, long‐term self‐renewal, and osteogenic induction of hiPSCs. After vitronectin (VN) peptide modification, the engineered surfaces facilitate adhesion, proliferation, colony formation, and the maintenance of pluripotency of hiPSCs up to passage 10 under fully defined conditions without Matrigel or protein coating. Further, this synthetic niche exhibits an appealing regulatory effect on the osteogenic conversion of hiPSCs to osteoblastic phenotype without an embryoid body formation step by co‐decoration of different ratios of VN and bone‐forming peptide. Such a well‐defined, xeno‐free 2D engineered microenvironment not only helps to accelerate the clinical development of hiPSCs, but also provides a safe and robust platform for the generation of osteoblast‐like cells or bone‐like tissues at different maturation levels. Thus, the strategy may hold great potential for application in cell therapy and bone tissue engineering.     

Discovery and Evaluation of a Functional Ternary Polymer Blend for Bone Repair: Translation from a Microarray to a Clinical Model

Skeletal tissue regeneration is often required following trauma, where substantial bone or cartilage loss may be encountered and is a significant driver for the development of biomaterials with a defined 3D structural network. Solvent blending is a process that avoids complications associated with conventional thermal or mechanical polymer blending or synthesis, opening up large areas of chemical and physical space, while potentially simplifying regulatory pathways towards in vivo application. Here ternary mixtures of natural and synthetic polymers were solvent blended and evaluated as potential bone tissue engineering matrices for osteoregeneration by the assessment of growth and differentiation of STRO‐1+ skeletal stem cells. Several of the blend materials were found to be excellent supports for human bone marrow‐derived STRO‐1+ skeletal cells and fetal skeletal cells, with the optimized blend exhibiting in vivo osteogenic potential, suggesting that these polymer blends could act as suitable matrices for bioengineering of hard tissues.     

Injectable Stem Cell‐Laden Photocrosslinkable Microspheres Fabricated Using Microfluidics for Rapid Generation of Osteogenic Tissue Constructs

Direct injection is a minimally invasive method of stem cell transplantation for numerous injuries and diseases. However, despite its promising potential, its clinical translation is difficult due to the low cell retention and engraftment after injection. With high versatility, high‐resolution control and injectability, microfabrication of stem‐cell laden biomedical hydrogels holds great potential as minimally invasive technology. Herein, a strategy of microfluidics‐assisted technology entrapping bone marrow‐derived mesenchymal stem cells (BMSCs) and growth factors in photocrosslinkable gelatin (GelMA) microspheres to ultimately generate injectable osteogenic tissue constructs is presented. Additionally, it is demonstrated that the GelMA microspheres can sustain stem cell viability, support cell spreading inside the microspheres and migration from the interior to the surface as well as enhance cell proliferation. This finding shows that encapsulated cells have the potential to directly and actively participate in the regeneration process. Furthermore, it is found that BMSCs encapsulated in GelMA microspheres show enhanced osteogenesis in vitro and in vivo, associated with a significant increase in mineralization. In short, the proposed strategy can be utilized to facilitate bone regeneration with minimum invasiveness, and can potentially be applied along with other matrices for extended applications.     

Cell‐Tethered Ligands Modulate Bone Remodeling by Osteoblasts and Osteoclasts

The goals of the present study are to establish an in vitro co‐culture model of osteoblast and osteoclast function and to quantify the resulting bone remodeling. The bone is tissue engineered using well‐defined silk protein biomaterials in 2D and 3D formats in combination with human cells. Parathyroid hormone (PTH) and glucose‐dependent insulinotropic peptide (GIP) are selected because of their roles in bone remodeling for expression in tethered format on human mesenchymal stem cells (hMSCs). The cell‐modified biomaterial surfaces are reconstructed from scanning electron microscopy images into 3D models for quantitative measurement of surface characteristics. Increased calcium deposition and surface roughness are found in 3D surface models of silk protein films remodeled by co‐cultures containing tethered PTH, and decreased surface roughness is found for the films remodeled by tethered GIP co‐cultures. Increased surface roughness is not found in monocultures of hMSCs expressing tethered PTH, suggesting that osteoclast‐osteoblast interactions in the presence of PTH signaling are responsible for the increased mineralization. These data point towards the design of in vitro bone models in which osteoblast‐osteoclast interactions are mimicked for a better understanding of bone remodeling.     

Anisotropic Nanoscale Presentation of Cell Adhesion Ligand Enhances the Recruitment of Diverse Integrins in Adhesion Structures and Mechanosensing‐Dependent Differentiation of Stem Cells

The nanoscale anisotropic patterns of bioactive ligands in the extracellular matrix regulate cell adhesion behaviors. However, the mechanisms of such regulation remain unclear. Here, RGD‐bearing gold nanorods (AuNRs) are conjugated with different aspect ratios (ARs, from 1 to 7) on cell culture substrates to decouple the effect of nanoscale anisotropic presentation of cell adhesive RGD peptides on cell adhesion. Compared with AuNRs with small ARs, AuNRs with large ARs significantly promote cell spreading, the alignment of the basal cytoskeletal structure, and nanopodia attachment. Furthermore, both ‐β3 and ‐β1 class integrins are recruited to AuNRs with large ARs, thereby promoting the development of focal adhesion toward fibrillar adhesion, whereas the recruitment of diverse integrins and the development of cell adhesion structures are hindered by small ARs AuNRs. The anisotropic presentation of ligands by large AR AuNRs better activates mechanotransduction signaling molecules. These findings are confirmed both in vitro and in vivo. Hence the enhanced mechanotransduction promotes osteogenic differentiation in stem cells. These findings demonstrate the potential use of well‐controlled synthetic nanoplatforms to unravel the fundamental mechanisms of cell adhesion and associated signaling at the molecular level and to provide valuable guidance for the rational design of biomaterials with tailored bioactive functions.     

Functionally Graded,Bone‐ and Tendon‐Like Polyurethane for Rotator Cuff Repair

Critical considerations in engineering biomaterials for rotator cuff repair include bone‐tendon‐like mechanical properties to support physiological loading and biophysicochemical attributes that stabilize the repair site over the long‐term. In this study, UV‐crosslinkable polyurethane based on quadrol (Q), hexamethylene diisocyante (H), and methacrylic anhydride (M; QHM polymers), which are free of solvent, catalyst, and photoinitiator, is developed. Mechanical characterization studies demonstrate that QHM polymers possesses phototunable bone‐ and tendon‐like tensile and compressive properties (12–74 MPa tensile strength, 0.6–2.7 GPa tensile modulus, 58–121 MPa compressive strength, and 1.5–3.0 GPa compressive modulus), including the capability to withstand 10 000 cycles of physiological tensile loading and reduce stress concentrations via stiffness gradients. Biophysicochemical studies demonstrate that QHM polymers have clinically favorable attributes vital to rotator cuff repair stability, including slow degradation profiles (5–30% mass loss after 8 weeks) with little‐to‐no cytotoxicity in vitro, exceptional suture retention ex vivo (2.79–3.56‐fold less suture migration relative to a clinically available graft), and competent tensile properties (similar ultimate load but higher normalized tensile stiffness relative to a clinically available graft) as well as good biocompatibility for augmenting rat supraspinatus tendon repair in vivo. This work demonstrates functionally graded, bone‐tendon‐like biomaterials for interfacial tissue engineering.     

Phase‐Transited Lysozyme as a Universal Route to Bioactive Hydroxyapatite Crystalline Film

A key factor for successful design of bioactive complex, organic–inorganic hybrid biomaterials is the facilitation and control of adhesion at interfaces, as many current synthetic biomaterials are inert, lacking interfacial bioactivity. In this regard, the development of a simple, unified way to biofunctionalize diverse organic and inorganic materials toward biomineralization remains a critical challenge. In this report, a universal biomimetic mineralization route that can be applied to virtually any type and morphology of scaffold materials is provided to induce nucleation and growth of hydroxyapatite (HAp) crystals based on phase‐transited lysozyme (PTL) coating. Surface‐anchored abundant functional groups in the PTL enrich the interface with strongly bonded calcium ions, facilitating the formation of HAp crystals in simulated body fluid with the morphology and alignment being similar to that observed in natural HAp in mineralized tissues. By the adhesion of amyloid contained in the PTL, such protein assembly could readily integrate HAp on ceramics, metals, semiconductors, and synthetic polymers irrespective of their size and morphology, with robust bonding stability and corresponding ultralow wear extent under normal bone pressure. This strategy successfully improves the in vivo osteoconductivity of Ti‐based implant, underpinning the expectation for such biomaterial in future biointerface and tissue engineering.     

Piezoelectric Nanotopography Induced Neuron‐Like Differentiation of Stem Cells

The biophysical characteristics of the extracellular matrix, such as nanotopography and bioelectricity, have a profound influence on cell proliferation, adhesion, differentiation, etc. Recognition of the function of a certain biophysical cue and fabrication of biomaterial scaffolds with specific properties would have important implications and significant applications in tissue engineering. Herein, nanotopographic and piezoelectric biomaterials are fabricated and the combination effect of and individual contribution to proliferation, adhesion, and neuron‐like differentiation of rat bone marrow‐derived mesenchymal stem cells (rbMSCs) are clarified via nanotopography and piezoelectricity. Piezoelectric polyvinylidene fluoride with nanostripe array structures is fabricated, which can generate a surface piezoelectric potential up to millivolt by cell movement and traction. The results reveal a more favorable effect on neuron‐like differentiation of rbMSCs from the combination of piezoelectricity and nanotopography rather than nanotopography alone, whereas nanotopography can increase cellular adhesion. This research provides a new insight into designing biomaterials for the potential application in neural tissue engineering.     

Self‐Supporting Graphene Hydrogel Film as an Experimental Platform to Evaluate the Potential of Graphene for Bone Regeneration

Graphene, a two dimensional carbonaceous material possessing a range of extraordinary properties, is considered promising for biomedical applications. Here, a simple form of graphene‐based bulk material–self‐supporting graphene hydrogel (SGH) film is used as a suitable platform to study the intrinsic properties of graphene both in vitro and in vivo. The free‐standing film show good cell adhesion, spreading, and proliferation. Films are implanted into subcutaneous sites of rats, and produce minimal fibrous capsule formation, and mild host tissue response in vivo. New blood vessel formation is also seen. The films swell and cracked in vivo, indicating the beginning of degradation. Of particular interest is that the film alone is found to be able to stimulate osteogenic differentiation of stem cells, without additional inducer, both in vitro and in vivo. Thus, this SGH film appears to be highly biocompatible and osteoinductive, demonstrating graphene's potential for bone regenerative medicine.     

“Tissue Papers” from Organ‐Specific Decellularized Extracellular Matrices

Using an innovative, tissue‐independent approach to decellularized tissue processing and biomaterial fabrication, the development of a series of “tissue papers” derived from native porcine tissues/organs (heart, kidney, liver, muscle), native bovine tissue/organ (ovary and uterus), and purified bovine Achilles tendon collagen as a control from decellularized extracellular matrix particle ink suspensions cast into molds is described. Each tissue paper type has distinct microstructural characteristics as well as physical and mechanical properties, is capable of absorbing up to 300% of its own weight in liquid, and remains mechanically robust (E = 1–18 MPa) when hydrated; permitting it to be cut, rolled, folded, and sutured, as needed. In vitro characterization with human mesenchymal stem cells reveals that all tissue paper types support cell adhesion, viability, and proliferation over four weeks. Ovarian tissue papers support mouse ovarian follicle adhesion, viability, and health in vitro, as well as support, and maintain the viability and hormonal function of nonhuman primate and human follicle‐containing, live ovarian cortical tissues ex vivo for eight weeks postmortem. “Tissue papers” can be further augmented with additional synthetic and natural biomaterials, as well as integrated with recently developed, advanced 3D‐printable biomaterials, providing a versatile platform for future multi‐biomaterial construct manufacturing.     

Injection and Self‐Assembly of Bioinspired Stem Cell‐Laden Gelatin/Hyaluronic Acid Hybrid Microgels Promote Cartilage Repair In Vivo

In this paper, a novel bioinspired stem cell‐laden microgel and related in vivo cartilage repair strategy are proposed. In particular, herein the preparation of new stem cell‐laden microgels, which can be injected into the chondral defect site in a minimally invasive way, and more importantly, capable of in situ self‐assembly into 3D macroporous scaffold without external stimuli, is presented. Specifically, thiolated gelatin (Gel‐SH) and vinyl sulfonated hyaluronic acid (HA‐VS) are first synthesized, and then stem cell‐laden gelatin/hyaluronic acid hybrid microgels (Gel‐HA) are generated by mixing Gel‐SH, HA‐VS, and bone mesenchymal stem cells (BMSCs) together via droplet‐based microfluidic approach, followed by gelation through fast and efficient thiol‐Michael addition reaction. The encapsulated BMSCs show high viability, proliferation, and chondrogenic differentiation potential in the microgels. Moreover, the in vitro test proves that BMSC‐laden Gel‐HA microgels are injectable without sacrificing BMSC viability, and more importantly, can self‐assemble into cartilage‐like scaffolds via cell–cell interconnectivity. In vivo experiments further confirm that the self‐assembled microgels can inhibit vascularization and hypertrophy. The Gel‐HA microgels and relevant cartilage repair strategy, i.e., injecting BMSC‐laden microgels separately and reconstructing chondral defect structure by microgel self‐assembly, provides a simple and effective method for cartilage tissue engineering and regenerative medicine.     

Channeled β‐TCP Scaffolds Promoted Vascularization and Bone Augmentation in Mandible of Beagle Dogs

A major hindrance to successful alveolar bone augmentation and ridge preservation using synthetic scaffolds is insufficient vascularization in the implanted bone grafts. The slow ingrowth of host vasculature from the bone bed of alveolar bone to the top of the implanted bone grafts leads to limited bone formation in the upper layers of the implanted grafts, which hinders the subsequent implantation of titanium dental implants. In this study, macroporous beta‐tricalcium phosphate (β‐TCP) scaffolds with multiple vertical hollow channels are fabricated that play a similar role as blood vessels for nutrient diffusion and cell migration. The results show that the hollow channels accelerate the degradation rate of the β‐TCP scaffolds and the in vitro release of a bone forming peptide‐1, which significantly promote proliferation and osteogenesis of human bone mesenchymal stem cells on the channeled scaffolds, compared to nonchanneled scaffolds in vitro. More volume of newly formed bone tissues with more blood vessels are augmented in the channeled scaffolds when implanted in mandibular bone defects of beagle dogs. Channeled scaffolds significantly promote new bone formation and augment the height of the mandible. These findings indicate channeled scaffolds facilitate vascularization and bone formation and have great potential for vascularized bone augmentation.     

Biological Assemblies Provide Novel Templates for the Synthesis of Biocomposites and Facilitate Cell Adhesion

Mechanical mismatch and the lack of interactions between implants and the natural tissue environment are major drawbacks in bone tissue engineering. Biomaterials mimicking the self‐assembly process and the composition of the bone matrix should provide new routes for fabricating biomaterials possessing novel osteoconductive and osteoinductive properties for bone repair. In the present study, bioinspired strategies are employed to design de novo self‐assembled chimeric protein hydrogels comprising leucine zipper motifs flanking a dentin matrix protein 1 domain, which is characterized as a mineralization nucleator. Results show that this chimeric protein could function as a hydroxyapatite nucleator in pseudo‐physiological buffer with the formation of highly oriented apatites similar to biogenic bone mineral. It could also function as an inductive substrate for osteoblast adhesion, promote cell surface integrin presentation and clustering, and modulate the formation of focal contacts. Such biomimetic “bottom‐up” construction with dual osteoconductive and osteoinductive properties should open new avenues for bone tissue engineering.     

Creating a Living Hyaline Cartilage Graft Free from Non‐Cartilaginous Constituents: An Intermediate Role of a Biomaterial Scaffold

A novel living hyaline cartilage graft (LhCG) with controllable dimensions and free of non‐cartilaginous constituents for articular regeneration is developed. As a living graft for regenerative medicine, LhCG is purely living tissue based and truly scaffold‐free. The process of neotissue formation in LhCG is mediated by an interim biomaterial‐based novel scaffolding system. This design highlights a philosophy of using biomaterials in engineered regenerative medicine as a transient guiding facility rather than a permanent part of substitute. The fabrication is designed and practiced in a continuous and integrated process, which attributes to its simplicity in operation. Because of the intrinsic non‐cell‐adhesive property of hydrogel scaffolds, articular chondrocytes’ phenotype is always preserved throughout the whole procedure, which has been tested and approved both in vitro and in vivo. In situ grafting trials in a rabbit model showcase high success rates in both cartilage repair and graft‐host integration. Beyond cartilage repair, this LhCG model may provide a living‐tissue‐based open platform or niche for multi‐tissue regenerations.     

Ice‐Templated Protein Nanoridges Induce Bone Tissue Formation

Little is known about the role of biocompatible protein nanoridges in directing stem cell fate and tissue regeneration due to the difficulty in forming protein nanoridges. Here an ice‐templating approach is proposed to produce semi‐parallel pure silk protein nanoridges. The key to this approach is that water droplets formed in the protein films are frozen into ice crystals (removed later by sublimation), pushing the surrounding protein molecules to be assembled into nanoridges. Unlike the flat protein films, the unique protein nanoridges can induce the differentiation of human mesenchymal stem cells (MSCs) into osteoblasts without any additional inducers, as well as the formation of bone tissue in a subcutaneous rat model even when not seeded with MSCs. Moreover, the nanoridged films induce less inflammatory infiltration than the flat films in vivo. This work indicates that decorating biomaterials surfaces with protein nanoridges can enhance bone tissue formation in bone repair.     

Bioinspired Development of an In Vitro Engineered Fracture Callus for the Treatment of Critical Long Bone Defects

Cell-based regenerative constructs provide hope for the restoration of tissue function in compromised biological conditions such as complex bone defects. A strategy mimicking the cascade of events of postnatal fracture healing suggests an implant design where progenitor cells provide the driving force for the construct's tissue forming capacity, while framing biomaterials provide cells with 3D cues to direct cellular processes. Large bone defects mainly heal through the formation of an intermediate endochondral fracture callus. The authors aimed to develop an in vitro engineered fracture callus manufactured by bioprinting to provide a spatially organized tissue construct based on: i) in vitro 3D primed human periosteum derived cells and ii) biocompatible thiol-ene alginate hydrogels, mimicking the cells and extracellular matrix present in the different zones of the callus. Cell viability and maintained osteochondrogenic differentiation upon bioprinting is confirmed in vitro. In vivo assessment displays that the developed biomaterials provided essential 3D cues that further guided the cells in their tissue forming process in the absence of additional stimulatory molecules. The reported findings confirm the appeal of a biomimetic approach to steer tissue development of in vitro engineered constructs and illustrate the suitability of bioprinting methodologies for the fabrication of living regenerative implants.     

Electrosprayed Enzyme Coatings as Bioinspired Alternatives to Bioceramic Coatings for Orthopedic and Oral Implants

The biological performance of orthopedic and oral implants can be significantly improved by functionalizing the non‐physiological metallic implant surface through the application of biologically active coatings. In this paper, a cost‐effective alternative to traditional biomedical coatings for bone substitution through exploitation of the specific advantages of the electrospray deposition technique for the immobilization of the enzyme alkaline phosphatase (ALP) onto the implant surface is presented. Since ALP increases the local inorganic phosphate concentration required for physiological mineralization of hard tissues, ALP coatings will enable enzyme‐mediated mineralization onto titanium surfaces. To evaluate the bone‐bioactive capacity of the ALP‐coated titanium surface, soaking experiments are performed. Although the purely inorganic so‐called simulated body fluid is the standard in vitro procedure for predictive studies on potential bone bonding in vivo, an alternative testing solution is proposed that also contains organic phosphates (cell culture medium supplemented with the organic β‐b;‐glycerophosphate (β‐b;‐GP) and serum proteins), thereby resembling the in vivo conditions more closely. Under these physiological conditions, the electrosprayed ALP coatings accelerated mineralization onto the titanium surface as compared to noncoated implant material by means of enzymatic pathways. Therefore, this novel approach toward implant fixation holds significant promise.     

Simulation of Cortical and Cancellous Bone to Accelerate Tissue Regeneration

Different tissues have complex anisotropic structures to support biological functions. Mimicking these complex structures in vitro remains a challenge in biomaterials designs. Here, inspired by different types of silk nanofibers, a composite materials strategy is pursued toward this challenge. A combination of fabrication methods is utilized to achieve separate control of amorphous and beta-sheet rich silk nanofibers in the same solution. Aqueous solutions containing two types of silk nanofibers are simultaneously treated with an electric field and with ethylene glycol diglycidyl ether (EGDE). Under these conditions, the beta-sheet rich silk nanofibers in the mixture responded to the electric field while the amorphous nanofibers are active in the crosslinking process with the EGDE. As a result, cryogels with anisotropic structures are prepared, including mimics for cortical- and cancellous-like bone biomaterials as a complex osteoinductive niche. In vitro studies revealed that mechanical cues of the cryogels induced osteodifferentiation of stem cells while the anisotropy inside the cryogels influenced immune reactions of macrophages. These bioactive cryogels also stimulated improved bone regeneration in vivo through modulation of inflammation, angiogenesis and osteogenesis responses, suggesting an effective strategy to develop bioactive matrices with complex anisotropic structures beneficial to tissue regeneration.