Fabrication of soft tissue engineering scaffolds by means of rapid prototyping techniques

Scaffolds are of great importance for tissue engineering because they enable the production of functional living implants out of cells obtained from cell culture. These scaffolds require individual external shape and well defined internal structure with interconnected porosity. The problem of the fabrication of prototypes from computer assisted design (CAD) data is well known in automotive industry. Rapid prototyping (RP) techniques are able to produce such parts. Some RP techniques exist for hard tissue implants. Soft tissue scaffolds need a hydrogel material. No biofunctional and cell compatible processing for hydrogels exists in the area of RP. Therefore, a new rapid prototyping (RP) technology was developed at the Freiburg Materials Research Center to meet the demands for desktop fabrication of hydrogels. A key feature of this RP technology is the three-dimensional dispensing of liquids and pastes in liquid media. The porosity of the scaffold is calculated and an example of the data conversion from a volume model to the plotting path control is demonstrated. The versatile applications of the new hydrogel scaffolds are discussed, including especially its potential for tissue engineering.     

Multifunctional chondroitin sulphate for cartilage tissue-biomaterial integration

A biologically active, high-strength tissue adhesive is needed for numerous medical applications in tissue engineering and regenerative medicine. Integration of biomaterials or implants with surrounding native tissue is crucial for both immediate functionality and long-term performance of the tissue. Here, we use the biopolymer chondroitin sulphate (CS), one of the major components of cartilage extracellular matrix, to develop a novel bioadhesive that is readily applied and acts quickly. CS was chemically functionalized with methacrylate and aldehyde groups on the polysaccharide backbone to chemically bridge biomaterials and tissue proteins via a twofold covalent link. Three-dimensional hydrogels (with and without cells) bonded to articular cartilage defects. In in vitro and in vivo functional studies this approach led to mechanical stability of the hydrogel and tissue repair in cartilage defects.     

Macroporous hydrogels based on 2-hydroxyethyl methacrylate. Part 6: 3D hydrogels with positive and negative surface charges and polyelectrolyte complexes in spinal cord injury repair

Macroporous hydrogels are artificial biomaterials commonly used in tissue engineering, including central nervous system (CNS) repair. Their physical properties may be modified to improve their adhesion properties and promote tissue regeneration. We implanted four types of hydrogels based on 2-hydroxyethyl methacrylate (HEMA) with different surface charges inside a spinal cord hemisection cavity at the Th8 level in rats. The spinal cords were processed 1 and 6 months after implantation and histologically evaluated. Connective tissue deposition was most abundant in the hydrogels with positively-charged functional groups. Axonal regeneration was promoted in hydrogels carrying charged functional groups; hydrogels with positively charged functional groups showed increased axonal ingrowth into the central parts of the implant. Few astrocytes grew into the hydrogels. Our study shows that HEMA-based hydrogels carrying charged functional groups improve axonal ingrowth inside the implants compared to implants without any charge. Further, positively charged functional groups promote connective tissue infiltration and extended axonal regeneration inside a hydrogel bridge.     

Magneto-Driven Gradients of Diamagnetic Objects for Engineering Complex Tissues

Engineering complex tissues represents an extraordinary challenge and, to date, there have been few strategies developed that can easily recapitulate native-like cell and biofactor gradients in 3D materials. This is true despite the fact that mimicry of these gradients may be essential for the functionality of engineered graft tissues. Here, a non-traditional magnetics-based approach is developed to predictably position naturally diamagnetic objects in 3D hydrogels. Rather than magnetizing the objects within the hydrogel, the magnetic susceptibility of the surrounding hydrogel precursor solution is enhanced. In this way, a range of diamagnetic objects (e.g., polystyrene beads, drug delivery microcapsules, and living cells) are patterned in response to a brief exposure to a magnetic field. Upon photo-crosslinking the hydrogel precursor, object positioning is maintained, and the magnetic contrast agent diffuses out of the hydrogel, supporting long-term construct viability. This approach is applied to engineer cartilage constructs with a depth-dependent cellularity mirroring that of native tissue. These are thought to be the first results showing that magnetically unaltered cells can be magneto-patterned in hydrogels and cultured to generate heterogeneous tissues. This work provides a foundation for the formation of opposing magnetic-susceptibility-based gradients within a single continuous material.     

Rapid Biofilm Eradication on Bone Implants Using Red Phosphorus and Near‐Infrared Light

Bone‐implant‐associated infections are common after orthopedic surgery due to impaired host immune response around the implants. In particular, when a biofilm develops, the immune system and antibiotic treatment find it difficult to eradicate, which sometimes requires a second operation to replace the infected implants. Most strategies have been designed to prevent biofilms from forming on the surface of bone implants, but these strategies cannot eliminate the biofilm when it has been established in vivo. To address this issue, a nonsurgical, noninvasive treatment for biofilm infection must be developed. Herein, a red‐phosphorus–IR780–arginine–glycine–aspartic‐acid–cysteine coating on titanium bone implants is prepared. The red phosphorus has great biocompatibility and exhibits efficient photothermal ability. The temperature sensitivity of Staphylococcus aureus biofilm is enhanced in the presence of singlet oxygen (¹O₂) produced by IR780. Without damaging the normal tissue, the biofilm can be eradicated through a safe near‐infrared (808 nm) photothermal therapy at 50 °C in vitro and in vivo. This approach reaches an antibacterial efficiency of 96.2% in vivo with 10 min of irradiation at 50 °C. Meanwhile, arginine–glycine–aspartic‐acid–cysteine decorated on the surface of the implant can improve the cell adhesion, proliferation, and osteogenic differentiation.     

25th Anniversary Article: Engineering Hydrogels for Biofabrication

With advances in tissue engineering, the possibility of regenerating injured tissue or failing organs has become a realistic prospect for the first time in medical history. Tissue engineering – the combination of bioactive materials with cells to generate engineered constructs that functionally replace lost and/or damaged tissue – is a major strategy to achieve this goal. One facet of tissue engineering is biofabrication, where three‐dimensional tissue‐like structures composed of biomaterials and cells in a single manufacturing procedure are generated. Cell‐laden hydrogels are commonly used in biofabrication and are termed “bioinks”. Hydrogels are particularly attractive for biofabrication as they recapitulate several features of the natural extracellular matrix and allow cell encapsulation in a highly hydrated mechanically supportive three‐dimensional environment. Additionally, they allow for efficient and homogeneous cell seeding, can provide biologically‐relevant chemical and physical signals, and can be formed in various shapes and biomechanical characteristics. However, despite the progress made in modifying hydrogels for enhanced bioactivation, cell survival and tissue formation, little attention has so far been paid to optimize hydrogels for the physico‐chemical demands of the biofabrication process. The resulting lack of hydrogel bioinks have been identified as one major hurdle for a more rapid progress of the field. In this review we summarize and focus on the deposition process, the parameters and demands of hydrogels in biofabrication, with special attention to robotic dispensing as an approach that generates constructs of clinically relevant dimensions. We aim to highlight this current lack of effectual hydrogels within biofabrication and initiate new ideas and developments in the design and tailoring of hydrogels. The successful development of a “printable” hydrogel that supports cell adhesion, migration, and differentiation will significantly advance this exciting and promising approach for tissue engineering.     

Hydrogel Bioink Reinforcement for Additive Manufacturing: A Focused Review of Emerging Strategies

Bioprinting is an emerging approach for fabricating cell-laden 3D scaffolds via robotic deposition of cells and biomaterials into custom shapes and patterns to replicate complex tissue architectures. Bioprinting uses hydrogel solutions called bioinks as both cell carriers and structural components, requiring bioinks to be highly printable while providing a robust and cell-friendly microenvironment. Unfortunately, conventional hydrogel bioinks have not been able to meet these requirements and are mechanically weak due to their heterogeneously crosslinked networks and lack of energy dissipation mechanisms. Advanced bioink designs using various methods of dissipating mechanical energy are aimed at developing next-generation cellularized 3D scaffolds to mimic anatomical size, tissue architecture, and tissue-specific functions. These next-generation bioinks need to have high print fidelity and should provide a biocompatible microenvironment along with improved mechanical properties. To design these advanced bioink formulations, it is important to understand the structure–property–function relationships of hydrogel networks. By specifically leveraging biophysical and biochemical characteristics of hydrogel networks, high performance bioinks can be designed to control and direct cell functions. In this review article, current and emerging approaches in hydrogel design and bioink reinforcement techniques are critically evaluated. This bottom-up perspective provides a materials-centric approach to bioink design for 3D bioprinting.     

A Review of 3D Printing Technology for Medical Applications

Donor shortages for organ transplantations are a major clinical challenge worldwide. Potential risks that are inevitably encountered with traditional methods include complications, secondary injuries, and limited source donors. Three-dimensional (3D) printing technology holds the potential to solve these limitations; it can be used to rapidly manufacture personalized tissue engineering scaffolds, repair tissue defects in situ with cells, and even directly print tissue and organs. Such printed implants and organs not only perfectly match the patient’s damaged tissue, but can also have engineered material microstructures and cell arrangements to promote cell growth and differentiation. Thus, such implants allow the desired tissue repair to be achieved, and could eventually solve the donor-shortage problem. This review summarizes relevant studies and recent progress on four levels, introduces different types of biomedical materials, and discusses existing problems and development issues with 3D printing that are related to materials and to the construction of extracellular matrix in vitro for medical applications.     

Alginate hydrogel as a promising scaffold for dental-derived stem cells: an in vitro study

The objectives of this study were to: (1) develop an injectable and biodegradable scaffold based on oxidized alginate microbeads encapsulating periodontal ligament (PDLSCs) and gingival mesenchymal stem cells (GMSCs); and (2) investigate the stem cell viability, and osteogenic differentiation of the stem cells in vitro. Stem cells were encapsulated using alginate hydrogel. The stem cell viability, proliferation and differentiation to adipogenic and osteogenic tissues were studied. To investigate the expression of both adipogenesis and ontogenesis related genes, the RNA was extracted and RT-PCR was performed. The degradation behavior of hydrogel based on oxidized sodium alginate with different degrees of oxidation was studied in PBS at 37?°C as a function of time by monitoring the changes in weight loss. The swelling kinetics of alginate hydrogel was also investigated. The results showed that alginate is a promising candidate as a non-toxic scaffold for PDLSCs and GMSCs. It also has the ability to direct the differentiation of these stem cells to osteogenic and adipogenic tissues as compared to the control group in vitro. The encapsulated stem cells remained viable in vitro and both osteo-differentiated and adipo-differentiated after 4?weeks of culturing in the induction media. It was found that the degradation profile and swelling kinetics of alginate hydrogel strongly depends on the degree of oxidation showing its tunable chemistry and degradation rate. These findings demonstrate for the first time that immobilization of PDLSCs and GMSCs in the alginate microspheres provides a promising strategy for bone tissue engineering.     

Matrix generation within a macroporous non-degradable implant for osteochondral defects is not enhanced with partial enzymatic digestion of the surrounding tissue: evaluation in an in vivo rabbit model

Articular cartilage defects are a significant source of pain, have limited ability to heal, and can lead to the development of osteoarthritis. However, a surgical solution is not available. To tackle this clinical problem, non-degradable implants capable of carrying mechanical load immediately after implantation and for the duration of implantation, while integrating with the host tissue, may be viable option. But integration between articular cartilage and non-degradable implants is not well studied. Our objective was to assess the in vivo performance of a novel macroporous, nondegradable, polyvinyl alcohol construct. We hypothesized that matrix generation within the implant would be enhanced with partial digestion of the edges of articular cartilage. Our hypothesis was tested by randomizing an osteochondral defect created in the trochlea of 14 New Zealand white rabbits to treatment with: (i) collagenase or (ii) saline, prior to insertion of the implant. At 1 and 3-month post-operatively, the gross morphology and histologic appearance of the implants and the surrounding tissue were assessed. At 3 months, the mechanical properties of the implant were also quantified. Overall, the hydrogel implants performed favorably; at all time-points and in all groups the implants remained well fixed, did not cause inflammation or synovitis, and did not cause extensive damage to the opposing articular cartilage. Regardless of treatment with saline or collagenase, at 1 month post-operatively implants from both groups had a contiguous interface with adjacent cartilage and were populated with chondrocyte-like cells. At 3 months fibrous encapsulation of all implants was evident, there was no difference between area of aggrecan staining in the collagenase versus saline groups, and implant modulus was similar in both groups; leading us to reject our hypothesis. In summary, a porous PVA osteochondral implant remained well fixed in a short term in vivo osteochondral defect model; however, matrix generation within the implant was not enhanced with partial digestion of adjacent articular cartilage.     

Mechanically Defined Microenvironment Promotes Stabilization of Microvasculature,Which Correlates with the Enrichment of a Novel Piezo‐1+ Population of Circulating CD11b+/CD115+ Monocytes

Vascularization is a critical step in the restoration of cellular homeostasis. Several strategies including localized growth factor delivery, endothelial progenitor cells, genetically engineered cells, gene therapy, and prevascularized implants have been explored to promote revascularization. But, long‐term stabilization of newly induced vessels remains a challenge. It has been shown that fibroblasts and mesenchymal stem cells can stabilize newly induced vessels. However, whether an injected biomaterial alone can serve as an instructive environment for angiogenesis remains to be elucidated. It is reported here that appropriate vascular branching, and long‐term stabilization can be promoted simply by implanting a hydrogel with stiffness matching that of fibrin clot. A unique subpopulation of circulating CD11b⁺ myeloid and CD11b⁺/CD115⁺ monocytes that express the stretch activated cation channel Piezo‐1, which is enriched prominently in the clot‐like hydrogel, is identified. These findings offer evidence for a mechanobiology paradigm in angiogenesis involving an interplay between mechanosensitive circulating cells and mechanics of tissue microenvironment.     

Enhanced intratumoral uptake of quantum dots concealed within hydrogel nanoparticles

Effective nanomedical devices for tumor imaging and drug delivery are not yet available. In an attempt to construct a more functional device for tumor imaging, we have embedded quantum dots (which have poor circulatory behavior) within hydrogel nanoparticles made of poly-N-isopropylacrylamide. We found that the hydrogel encapsulated quantum dots are more readily taken up by cultured tumor cells. Furthermore, in a melanoma model, hydrogel encapsulated quantum dots also preferentially accumulate in the tumor tissue compared with normal tissue and have ～16-fold greater intratumoral uptake compared to non-derivatized quantum dots. Our results suggest that these derivatized quantum dots, which have greatly improved tumor localization, may enhance cancer monitoring and chemotherapy.     

Biodegradable Hydroxyapatite–Polymer Composites

The fracture of bone due to trauma or due to natural aging is one of the most frequent types of tissue failures. Treatment frequently requires the implantation of a temporary or permanent prosthesis. The implanted materials may include the components of artificial joints, plates, and screws for fracture fixation. Typically, such implants are intended only to provide structural support or to serve as templates for bone re‐growth. In general they are intended to remain in place for the life of the patient or to be removed in a second surgical procedure. This report provides a short review of the synthetic reconstructive bone implants. Following this, the rationale and basics of the biodegradable hydroxyapatite–polymer composite approach are described.     

Dual growth factor-releasing nanoparticle/hydrogel system for cartilage tissue engineering

In order to induce the chondrogenesis of mesenchymal stem cells (MSCs) in tissue engineering, a variety of growth factors have been adapted and encouraging results have been demonstrated. In this study, we developed a delivery system for dual growth factors using a gelation rate controllable alginate solution (containing BMP-7) and polyion complex nanoparticles (containing TGF-β₂) to be applied for the chondrogenesis of MSCs. The dual growth factors (BMP-7/TGF-β₂)-loaded nanoparticle/hydrogel system showed a controlled release of both growth factors: a faster release of BMP-7 and a slower release of TGF-β₂, ca., approximately 80 and 30% release at the end of an incubation period (21 days), respectively, which may be highly desirable for chondrogenic differentiation of MSCs. On the contrary, the release of each growth factor from the dual growth factors-loaded hydrogel (without the nanoparticles) was much slower than that of the nanoparticle/hydrogel system, approximately 36% (BMP-7) and 16% (TGF-β₂) for 21 days, and this is more than likely attributed to the aggregation between growth factors during the hydrogel fabrication step. The nanoparticle/hydrogel system with separate growth factor loading may provide desirable growth factor delivery kinetics for cartilage regeneration, as well as the chondrogenesis of MSCs.     

Identifying differentiation stage of individual primary hematopoietic cells from mouse bone marrow by multivariate analysis of TOF-secondary ion mass spectrometry data

The ability to self-renew and differentiate into multiple types of blood and immune cells renders hematopoietic stem and progenitor cells (HSPCs) valuable for clinical treatment of hematopoietic pathologies and as models of stem cell differentiation for tissue engineering applications. To study directed hematopoietic stem cell (HSC) differentiation and identify the conditions that recreate the native bone marrow environment, combinatorial biomaterials that exhibit lateral variations in chemical and mechanical properties are employed. New experimental approaches are needed to facilitate correlating cell differentiation stage with location in the culture system. We demonstrate that multivariate analysis of time-of-flight secondary ion mass spectrometry (TOF-SIMS) data can be used to identify the differentiation state of individual hematopoietic cells (HCs) isolated from mouse bone marrow. Here, we identify primary HCs from three distinct stages of B cell lymphopoiesis at the single cell level: HSPCs, common lymphoid progenitors, and mature B cells. The differentiation state of individual HCs in a test set could be identified with a partial least-squares discriminant analysis (PLS-DA) model that was constructed with calibration spectra from HCs of known differentiation status. The lowest error of identification was obtained when the intrapopulation spectral variation between the cells in the calibration and test sets was minimized. This approach complements the traditional methods that are used to identify HC differentiation stage. Further, the ability to gather mass spectrometry data from single HSCs cultured on graded biomaterial substrates may provide significant new insight into how HSPCs respond to extrinsic cues as well as the molecular changes that occur during cell differentiation.     

A cultured living bone equivalent enhances bone formation when compared to a cell seeding approach

The development of cell therapy methods to confer osteogenic potential to synthetic bone replacement materials has become common during the last years. At present, in the bone tissue engineering field, two different approaches use patient own cultured osteogenic cells in combination with a scaffold material to engineer autologous osteogenic grafts. One of the approaches consists of seeding cells on a suitable biomaterial, after which the construct is ready for implantation. In the other approach, the seeded cells are further cultured on the scaffold to obtain in vitro formed bone (extracellular matrix and cells), prior to implantation. In the present study, we investigated the in vivo osteogenic potential of both methods through the implantation of porous hydroxyapatite (HA) scaffolds coated with a layer of in vitro formed bone and porous HA scaffolds seeded with osteogenic cells. Results showed that as early as 2 days after implantation, de novo bone tissue was formed on scaffolds in which an in vitro bone-like tissue was cultured, while it was only detected on the cell seeded implants from 4 days onwards. In addition, after 4 days of implantation statistical analysis revealed a significantly higher amount of bone in the bone-like tissue containing scaffolds as compared to cell seeded ones.     

3D-Cultivation of bone marrow stromal cells on hydroxyapatite scaffolds fabricated by dispense-plotting and negative mould technique

The main principle of a bone tissue engineering (BTE) strategy is to cultivate osteogenic cells in an osteoconductive porous scaffold. Ceramic implants for osteogenesis are based mainly on hydroxyapatite (HA), since this is the inorganic component of bone. Rapid Prototyping (RP) is a new technology in research for producing ceramic scaffolds. This technology is particularly suitable for the fabrication of individually and specially tailored single implants. For tissue engineering these scaffolds are seeded with osteoblast or osteoblast precursor cells. To supply the cultured osteoblastic cells efficiently with nutrition in these 3D-geometries a bioreactor system can be used. The aim of this study was to analyse the influence of differently fabricated HA-scaffolds on bone marrow stromal cells. For this, two RP-techniques, dispense-plotting and a negative mould method, were used to produce porous ceramics. The manufactured HA-scaffolds were then cultivated in a dynamic system (bioreactor) with an osteoblastic precursor cell line. In our study, the applied RP-techniques give the opportunity to design and process HA-scaffolds with defined porosity, interconnectivity and 3D pore distribution. A higher differentiation of bone marrow stromal cells could be detected on the negative mould fabricated scaffolds, while cell proliferation was higher on the dispense-plotted scaffolds. Nevertheless, both scaffold types can be used in tissue engineering applications.     

Near‐Infrared Light‐Sensitive Polyvinyl Alcohol Hydrogel Photoresist for Spatiotemporal Control of Cell‐Instructive 3D Microenvironments

Advanced hydrogel systems that allow precise control of cells and their 3D microenvironments are needed in tissue engineering, disease modeling, and drug screening. Multiphoton lithography (MPL) allows true 3D microfabrication of complex objects, but its biological application requires a cell‐compatible hydrogel resist that is sufficiently photosensitive, cell‐degradable, and permissive to support 3D cell growth. Here, an extremely photosensitive cell‐responsive hydrogel composed of peptide‐crosslinked polyvinyl alcohol (PVA) is designed to expand the biological applications of MPL. PVA hydrogels are formed rapidly by ultraviolet light within 1 min in the presence of cells, providing fully synthetic matrices that are instructive for cell‐matrix remodeling, multicellular morphogenesis, and protease‐mediated cell invasion. By focusing a multiphoton laser into a cell‐laden PVA hydrogel, cell‐instructive extracellular cues are site‐specifically attached to the PVA matrix. Cell invasion is thus precisely guided in 3D with micrometer‐scale spatial resolution. This robust hydrogel enables, for the first time, ultrafast MPL of cell‐responsive synthetic matrices at writing speeds up to 50 mm s^?1. This approach should enable facile photochemical construction and manipulation of 3D cellular microenvironments with unprecedented flexibility and precision.     

Regulation of RAW 264.7 macrophages behavior on anodic TiO<Subscript>2</Subscript> nanotubular arrays

Titanium (Ti) implants with TiO₂ nanotubular arrays on the surface could regulate cells adhesion, proliferation and differentiation to determine the bone integration. Additionally, the regulation of immune cells could improve osteogenesis or lead in appropriate immune reaction. Thus, we evaluate the behavior of RAW264.7 macrophages on TiO₂ nanotubular arrays with a wide range diameter (from 20 to 120 nm) fabricated by an electrochemical anodization process. In this work, the proliferation, cell viability and cytokine/chemokine secretion were evaluated by CCK-8, live/dead staining and ELISA, respectively. SEM and confocal microscopy were used to observe the adhesion morphology. Results showed that the small size nanotube surface was benefit for the macrophages adhesion and proliferation, while larger size surface could reduce the inflammatory response. These findings contribute to the design of immune-regulating Ti implants surface that supports successful implantation.     

Strategies for creating living,additively manufactured,open-cellular metal and alloy implants by promoting osseointegration,osteoinduction and vascularization: An overview

Additive manufacturing of porous, open-cellular metal or alloy implants, fabricated by laser or electron beam melting of a powder bed, is briefly reviewed in relation to optimizing biomechanical compatibility by assuring elastic (Young’s) modulus matching of proximate bone, along with corresponding pore sizes assuring osseointegration and vasculature development and migration. In addition, associated, requisite compressive and fatigue strengths for such implants are described. Strategies for optimizing osteoblast (bone cell) development and osteoinduction as well as vascularization of tissue in 3D scaffolds and tissue engineering constructs for bone repair are reviewed in relation to the biology of osteogenesis and neovascularization in bone, and the role of associated growth factors, bone morphogenic proteins, signaling molecules and the like. Prospects for infusing hydrogel/collagen matrices containing these cellular and protein components or surgically extracted intramedullary (bone marrow) concentrate/aspirate containing these biological and cell components into porous implants are discussed, as strategies for creating living implants, which over the long term would act as metal or alloy scaffolds.