Direct 3D printing of decellularized matrix embedded composite polycaprolactone scaffolds for cartilage regeneration.

Treatment options for large osteochondral injuries (OCIs) are limited by donor tissue scarcity, morbidity, and anatomic mismatch. 3D printing technology can produce patient-specific scaffolds to address these large defects. Thermoplastics like polycaprolactone (PCL) offer necessary mechanical properties, but lack bioactivity. We fabricated 3D printed PCL scaffolds embedded with polylactic acid microspheres containing decellularized cartilage matrix (DM). DM incorporation within polylactic acid microspheres prevented its thermal degradation during the 3D printing process. The scaffolds replicated the mechanical properties of native cartilage and demonstrated controlled release of DM proteins. Human mesenchymal stem cells (hMSCs) seeded on the composite scaffolds with DM and cultured in basal media self-assembled into aggregates mimicking mesenchymal condensates during embryonic development. The DM composite scaffolds also induced higher expression of biochemical markers of cartilage development than controls, providing evidence for their translational application in the treatment of OCIs. The present study demonstrates the potential of direct incorporation of DM with thermoplastics for 3D printing of patient-specific scaffolds for osteochondral regeneration.

Elucidation of bio-inspired hydroxyapatie crystallization on oxygen-plasma modified 3D printed poly-caprolactone scaffolds.

Bioapatite formation in bones is a slow process starting with deposition of calcium phosphate and then its nucleation and crystallization into hydroxyapatite crystals. If the same process can be replicated on tissue engineered scaffolds, it will result in the formation of biomimetic bone constructs that will have comparable mechanical properties to native tissue. To mimic the same process on 3D printed polycaprolactone (PCL) scaffolds oxygen plasma treatment was performed to modify their surface chemistry. The attenuated total reflectance-fourier transform infrared (ATR-FTIR) analysis showed formation of carboxyl groups on the PCL surface with corresponding increase in roughness as analyzed by atomic force microscope (AFM) studies. A biomimetic acellular mineralization procedure was then utilized to deposit calcium minerals on these scaffolds. Though amorphous calcium phosphate was deposited on all the scaffolds with highest amount on PCL scaffolds with tricalcium phosphate (TCP), biomimetic hydroxyapatite crystals were only formed on oxygen plasma treated scaffolds, as shown by X-ray diffraction (XRD) analysis. The COOH groups on the plasma treated scaffolds acted as nucleation sites for amorphous calcium phosphate and the crystal growth was observed in the (211) plane simulating the crystal growth in developing bones. The ATR-FTIR study demonstrated the carbonated nature of these hydroxyapatite crystals mimicking that of bioapatite. The electronegative COOH groups mimic the negative amino acid side chains in collagen Type I present in bone tissue and the carbonated environment helps in creating bioapatite like deposits. The present study demonstrated the important role of PCL surface chemistry in mimicking a bone like mineralization process in vitro. This work details novel insights regarding improved mineralization of 3D printed PCL scaffolds useful for the development of more biomimetic bone constructs with improved mechanical properties.

Novel Process for 3D Printing Decellularized Matrices.

3D bioprinting aims to create custom scaffolds that are biologically active and accommodate the desired size and geometry. A thermoplastic backbone can provide mechanical stability similar to native tissue while biologic agents offer compositional cues to progenitor cells, leading to their migration, proliferation, and differentiation to reconstitute the original tissues/organs1 , 2. Unfortunately, many 3D printing compatible, bioresorbable polymers (such as polylactic acid, PLA) are printed at temperatures of 210 °C or higher - temperatures that are detrimental to biologics. On the other hand, polycaprolactone (PCL), a different type of polyester, is a bioresorbable, 3D printable material that has a gentler printing temperature of 65 °C. Therefore, it was hypothesized that decellularized extracellular matrix (DM) contained within a thermally protective PLA barrier could be printed within PCL filament and remain in its functional conformation. In this work, osteochondral repair was the application for which the hypothesis was tested. As such, porcine cartilage was decellularized and encapsulated in polylactic acid (PLA) microspheres which were then extruded with polycaprolactone (PCL) into filament to produce 3D constructs via fused deposition modeling. The constructs with or without the microspheres (PLA-DM/PCL and PCL(-), respectively) were evaluated for differences in surface features.

Microspheres containing decellularized cartilage induce chondrogenesis in vitro and remain functional after incorporation within a poly(caprolactone) filament useful for fabricating a 3D scaffold.

In this study, articular cartilage was decellularized preserving a majority of the inherent proteins, cytokines, growth factors and sGAGs. The decellularized cartilage matrix (dCM) was then encapsulated in poly(lactic acid) microspheres (MS + dCM) via double emulsion. Blank microspheres without dCM, MS(-), were also produced. The microspheres were spherical in shape and protein encapsulation efficiency within MS + dCM was 63.4%. The sustained release of proteins from MS + dCM was observed over 4 weeks in vitro. Both MS + dCM and MS(-) were cytocompatible. The sustained delivery of retained growth factors and cytokines from MS + dCM promoted cell migration in contrast to MS(-). Subsequently, chondrogenesis of human mesenchymal stem cells was upregulated in presence of MS + dCM as evidenced from immunohistochemistry, biochemical quantification and qPCR studies. Specifically, collagen II, aggrecan and SOX 9 gene expression were increased in the presence of MS + dCM by an order or more in magnitude compared to MS(-) with concomitant downregulation of hypertrophic genes (COL X) despite being cultured in the absence of chondrogenic media, (p < 0.05). Lastly, microspheres containing alkaline phosphatase (MS + ALP), a surrogate to assess the thermal stability of dCM proteins, incorporated within poly(caprolactone) filaments showed that the enzyme remained functional after filament production by melt extrusion. The establishment of a novel, thermally stable process for producing filaments containing chondroinductive microspheres provides evidence supporting subsequent development of a clinically-relevant, 3D scaffold fabricated from them for osteochondral regeneration and repair.