Osteochondral regenerative engineering: challenges, state-of-the-art and translational perspectives.

Despite quantum leaps, the biomimetic regeneration of cartilage and osteochondral regeneration remains a major challenge, owing to the complex and hierarchical nature of compositional, structural and functional properties. In this review, an account of the prevailing challenges in biomimicking the gradients in porous microstructure, cells and extracellular matrix (ECM) orientation is presented. Further, the spatial arrangement of the cues in inducing vascularization in the subchondral bone region while maintaining the avascular nature of the adjacent cartilage layer is highlighted. With rapid advancement in biomaterials science, biofabrication tools and strategies, the state-of-the-art in osteochondral regeneration since the last decade has expansively elaborated. This includes conventional and additive manufacturing of synthetic/natural/ECM-based biomaterials, tissue-specific/mesenchymal/progenitor cells, growth factors and/or signaling biomolecules. Beyond the laboratory-based research and development, the underlying challenges in translational research are also provided in a dedicated section. A new generation of biomaterial-based acellular scaffold systems with uncompromised biocompatibility and osteochondral regenerative capability is necessary to bridge the clinical demand and commercial supply. Encompassing the basic elements of osteochondral research, this review is believed to serve as a standalone guide for early career researchers, in expanding the research horizon to improve the quality of life of osteoarthritic patients affordably.

Injectable and biodegradable piezoelectric hydrogel for osteoarthritis treatment.

Osteoarthritis affects millions of people worldwide but current treatments using analgesics or anti-inflammatory drugs only alleviate symptoms of this disease. Here, we present an injectable, biodegradable piezoelectric hydrogel, made of short electrospun poly-L-lactic acid nanofibers embedded inside a collagen matrix, which can be injected into the joints and self-produce localized electrical cues under ultrasound activation to drive cartilage healing. In vitro, data shows that the piezoelectric hydrogel with ultrasound can enhance cell migration and induce stem cells to secrete TGF-ß1, which promotes chondrogenesis. In vivo, the rabbits with osteochondral critical-size defects receiving the ultrasound-activated piezoelectric hydrogel show increased subchondral bone formation, improved hyaline-cartilage structure, and good mechanical properties, close to healthy native cartilage. This piezoelectric hydrogel is not only useful for cartilage healing but also potentially applicable to other tissue regeneration, offering a significant impact on the field of regenerative tissue engineering.

Evaluation of implant properties, safety profile and clinical efficacy of patient-specific acrylic prosthesis in cranioplasty using 3D binderjet printed cranium model: A pilot study.

There exists a significant demand to develop patient-specific prosthesis in reconstruction of cranial vaults after decompressive craniectomy. we report here, the outcomes of an unicentric pilot study on acrylic cranial prosthesis fabricated using a 3D printed cranium model with its clinically relevant mechanical properties. METHODS: The semi-crystalline polymethyl methacrylate (PMMA) implants, shaped to the cranial defects of 3D printed cranium model, were implanted in 10 patients (mean age, 40.8 ± 14.8 years). A binderjet 3D printer was used to create patient-specific mould and PMMA was casted to fabricate prosthesis which was analyzed for microstructure and properties. Patients were followed up for allergy, infection and cosmesis for a period of 6 months. RESULTS: As-cast PMMA flap exhibited hardness of 15.8 ± 0.24Hv, tensile strength of 30.7 ± 3.9 MPa and elastic modulus of 1.5 ± 0.1 GPa. 3D microstructure of the semi-crystalline acrylic implant revealed 2.5-15 µm spherical isolated pores. The mean area of the calvarial defect in craniectomy patients was 94.7 ± 17.4 cm2. We achieved a cranial index of symmetry (CIS -%) of 94.5 ± 3.9, while the average post-operative Glasgow outcome scale (GOS) score recorded was 4.2 ± 0.9. CONCLUSIONS: 3D printing based patient-specific design and fabrication of acrylic cranioplasty implant is safe and achieves acceptable cosmetic and clinical outcomes in patients with decompressive craniectomy. Our study ensured clinically acceptable structural and mechanical properties of implanted PMMA, suggesting that a low cost 3D printer based PMMA flap is an affordable option for cranioplasty in resource constrained settings.

Acoustic Poration and Dynamic Healing of Mammalian Cell Membranes during Inkjet Printing.

We have investigated the effect of piezoelectric actuating voltage on cell behavior after drop on demand inkjet printing using mouse 3T3 cells as a model cell line. Cell viability after printing was assessed using a live/dead assay, Alamar Blue as an assay for cell proliferation, and propidium iodide (PI) and Texas Red labeled dextran molecular probes to assess cell membrane integrity. No significant difference was found for the cell death rate compared between an unprinted control population and after printing at 80, 90, and 100 V, respectively. However, cell proliferation was lower than that of the control population at all time points postprinting. Cell membrane integrity was quantified using PI and dextran probes of mean molecular weight of 3, 10, 40, and 70 kDa. Total membrane damage (assessed by PI) increased with increasing piezoelectric actuator driving voltage, and this was always greater than the unprinted control cells. The uptake of the labeled dextran only occurs after inkjet printing and was never observed with the control cells. The largest dextran molecular probe of 70 kDa was only taken up by cells after printing using the lower printing voltages of 80 and 90 V and was absent after printing at 100 V. At the two lower printing voltages, the membrane damage is recovered, and no dextran molecule penetrated the cells 2 h after printing. However, printing at 100 V leads to an increased uptake of 3 and 10 kDa dextran molecules, the retention of membrane porosity, and continued uptake of these 3 and 10 kDa dextran for greater than 2 h postprinting. We hypothesize that the change in membrane porosity with increasing actuation voltage can be explained by distinct nucleation and growth stages for pore formation in response to printing stress.

Probing Ink-Powder Interactions during 3D Binder Jet Printing Using Time-Resolved X-ray Imaging.

Capillary-driven ink infiltration through a porous powder bed in three-dimensional (3D) binder jet printing (inkjet printing onto a powder bed) controls the printing resolution and as-printed "green" strength of the resulting object. However, a full understanding of the factors controlling the kinetics of the infiltration remains incomplete. Here, high-resolution in situ synchrotron radiography provides time-resolved imaging of the penetration of an aqueous solution of eythylene glycol through a porous alumina powder bed, used as a model system. A static drop-on-demand inkjet printer was used to dispense liquid droplets onto a powder surface. The subsequent migration of the liquid front and its interactions with powder particles were tracked using fast synchrotron X-radiography in the Diamond Synchrotron, with phase-contrast imaging at a frame rate of 500 Hz. Image processing and analysis reveal that both the time-dependent increment in the wetting area and the propagation of the "interface leading edge" exhibit heterogeneous behavior in both temporal and spatial domains. However, mean infiltration kinetics are shown to be consistent with existing infiltration models based on the Washburn equation modified to account for the spreading of the liquid drop on the powder surface and using a modified term for the bed porosity.

3D inkjet printing of biomaterials with strength reliability and cytocompatibility: Quantitative process strategy for Ti-6Al-4V.

Among additive manufacturing (AM) techniques, laser or electron beam based processes have been widely investigated for metallic implants. Despite the potential in manufacturing of patient-specific biomedical implants, 3D inkjet powder printing (3DIJPP, a variant of AM) of biomaterials is still in its infancy, as little is known quantitatively about the transient process physics and dynamics. An equally important challenge has been the ink formulation to manufacture biomaterials with reliable mechanical properties and desired biocompatibility. We have developed, for the very first time, the theoretical foundation and experimental formulation of a unique process strategy involving the 'on-demand' delivery of a novel in situ polymerisable acrylic ink system to print a model biomaterial, Ti-6Al-4V. The post-ejection in-flight dynamics of ink droplets have been captured in situ by employing high speed stroboscopic shadowgraphy, to quantitatively estimate the dimensionless numbers of fluid physics for 'printability' assessment. Washburn model was adapted extensively to quantify the capillary ink infiltration time in porous powder bed of finite thickness. On the other hand, particle tracking mode in diffusing wave spectroscopy (DWS) was exploited to analyse the timescale for effective binding of powder particles during in situ polymerisation. The clinically relevant combination of 3D porous architecture with 98.4% interconnectivity among 10-40 µm pores together with modest combination of elastic modulus (4 GPa) and strength reliability (Weibull modulus ∼8.1) establish the potential of inkjet printed Ti-6Al-4V as cortical bone analogue. A better cell attachment, viability, cytoskeletal spreading with pronounced proliferation of murine fibroblasts and pre-osteoblasts on 3DIJPP Ti-6Al-4V, when benchmarked against the metallurgically processed (commercial) or selective laser melted (SLM) Ti-6Al-4V, has been demonstrated, in vitro. The enhanced cellular activities on the 3DIJPP Ti-6Al-4V was explained in terms of an interplay among the elastic stiffness, surface roughness and wettability against the same benchmarking. It is conceived that the quantitative understanding of the integrated process physics and dynamics to print Ti-6Al-4V with reliable mechanical properties together with better cytocompatibility can lead to a paradigm shift in adapting the scalable 3DIJPP for manufacturing of metallic biomaterials.

Microstructure and compression properties of 3D powder printed Ti-6Al-4V scaffolds with designed porosity: Experimental and computational analysis.

The osseointegration of metallic implants depends on an effective balance among designed porosity to facilitate angiogenesis, tissue in-growth and bone-mimicking elastic modulus with good strength properties. While addressing such twin requirements, the present study demonstrates a low temperature additive manufacturing based processing strategy to fabricate Ti-6Al-4V scaffolds with designed porosity using inkjet-based 3D powder printing (3DPP). A novel starch-based aqueous binder was prepared and the physico-chemical parameters such as pH, viscosity, and surface tension were optimized for drop-on-demand (DOD) based thermal inkjet printing. Micro-computed tomography (micro-CT) of sintered scaffolds revealed a 57% total porosity in homogeneously porous scaffold and 45% in the gradient porous scaffold with 99% interconnectivity among the micropores. Under uniaxial compression testing, the strength of homogeneously porous and gradient porous scaffolds were ~47MPa and ~90MPa, respectively. The progressive failure in homogeneously porous scaffold was recorded. In parallel to experimental measurements, finite element (FE) analyses have been performed to study the stress distribution globally and also locally around the designed pores. Consistent with FE analyses, a higher elastic modulus was recorded with gradient porous scaffolds (~3GPa) than the homogenously porous scaffolds (~2GPa). While comparing with the existing literature reports, the present work, for the first time, establishes 'direct powder printing methodology' of Ti-6Al-4V porous scaffolds with biomedically relevant microstructural and mechanical properties. Also, a new FE analysis approach, based on the critical understanding of the porous architecture using micro-CT results, is presented to realistically predict the compression response of porous scaffolds.