Development of photoreactive demineralized bone matrix 3D printing colloidal inks for bone tissue engineering.

Demineralized bone matrix (DBM) has been widely used clinically for dental, craniofacial and skeletal bone repair, as an osteoinductive and osteoconductive material. 3D printing (3DP) enables the creation of bone tissue engineering scaffolds with complex geometries and porosity. Photoreactive methacryloylated gelatin nanoparticles (GNP-MAs) 3DP inks have been developed, which display gel-like behavior for high print fidelity and are capable of post-printing photocrosslinking for control of scaffold swelling and degradation. Here, novel DBM nanoparticles (DBM-NPs, ∼400 nm) were fabricated and characterized prior to incorporation in 3DP inks. The objectives of this study were to determine how these DBM-NPs would influence the printability of composite colloidal 3DP inks, assess the impact of ultraviolet (UV) crosslinking on 3DP scaffold swelling and degradation and evaluate the osteogenic potential of DBM-NP-containing composite colloidal scaffolds. The addition of methacryloylated DBM-NPs (DBM-NP-MAs) to composite colloidal inks (100:0, 95:5 and 75:25 GNP-MA:DBM-NP-MA) did not significantly impact the rheological properties associated with printability, such as viscosity and shear recovery or photocrosslinking. UV crosslinking with a UV dosage of 3 J/cm2 directly impacted the rate of 3DP scaffold swelling for all GNP-MA:DBM-NP-MA ratios with an ∼40% greater increase in scaffold area and pore area in uncrosslinked versus photocrosslinked scaffolds over 21 days in phosphate-buffered saline (PBS). Likewise, degradation (hydrolytic and enzymatic) over 21 days for all DBM-NP-MA content groups was significantly decreased, ∼45% less in PBS and collagenase-containing PBS, in UV-crosslinked versus uncrosslinked groups. The incorporation of DBM-NP-MAs into scaffolds decreased mass loss compared to GNP-MA-only scaffolds during collagenase degradation. An in vitro osteogenic study with bone marrow-derived mesenchymal stem cells demonstrated osteoconductive properties of 3DP scaffolds for the DBM-NP-MA contents examined. The creation of photoreactive DBM-NP-MAs and their application in 3DP provide a platform for the development of ECM-derived colloidal materials and tailored control of biochemical cue presentation with broad tissue engineering applications.

Extracellular matrix component-derived nanoparticles for drug delivery and tissue engineering.

The extracellular matrix (ECM) consists of a complex combination of proteins, proteoglycans, and other biomolecules. ECM-based materials have been demonstrated to have high biocompatibility and bioactivity, which may be harnessed for drug delivery and tissue engineering applications. Herein, nanoparticles incorporating ECM-based materials and their applications in drug delivery and tissue engineering are reviewed. Proteins such as gelatin, collagen, and fibrin as well as glycosaminoglycans including hyaluronic acid, chondroitin sulfate, and heparin have been employed for cancer therapeutic delivery, gene delivery, and wound healing and regenerative medicine. Strategies for modifying and functionalizing these materials with synthetic and natural polymers or to enable stimuli-responsive degradation and drug release have increased the efficacy of these materials and nano-systems. The incorporation and modification of ECM-based materials may be used to drive drug targeting and increase tissue-specific cell differentiation more effectively.

Aspects of a Suspended Bioprinting System Affect Cell Viability and Support Bath Properties.

Suspended hydrogel printing is a growing method for fabricating bioprinted hydrogel constructs, largely due to how it enables nonviscous hydrogel inks to be used in extrusion printing. In this work, a previously developed poly(N-isopropylacrylamide)-based thermogelling suspended bioprinting system was examined in the context of chondrocyte-laden printing. Material factors such as ink concentration and cell concentration were found to have a significant effect on printed chondrocyte viability. In addition, the heated poloxamer support bath was able to maintain chondrocyte viability for up to 6 h of residence within the bath. The relationship between the ink and support bath was also assessed by measuring the rheological properties of the bath before and after printing. Bath storage modulus and yield stress decreased during printing as nozzle size was reduced, indicating the likelihood that dilution occurs over time through osmotic exchange with the ink. Altogether this work demonstrates the promise for printing high-resolution cell-encapsulating tissue engineering constructs, while also elucidating complex relationships between the ink and bath, which must be taken into consideration when designing suspended printing systems.

Bioinspired electrospun decellularized extracellular matrix scaffolds promote muscle regeneration in a rat skeletal muscle defect model.

Volumetric muscle loss is a debilitating injury that can leave patients with long-lasting or permanent structural and functional deficits. With clinical treatments failing to address these shortcomings, there is a great need for tissue-engineered therapies to promote skeletal muscle regeneration. In this study, we aim to assess the potential for electrospun decellularized skeletal muscle extracellular matrix (dECM) to promote skeletal muscle regeneration in a rat partial thickness tibialis anterior defect model. Aligned electrospun scaffolds with varying degrees of crosslinking density were implanted into the defect site and compared to an empty defect control. After 8 weeks, muscles were harvested, weighed, and cellular and morphological analyses were performed via histology and immunohistochemistry. Cell infiltration, angiogenesis, and myogenesis were observed in the defect site in both dECM groups. However, favorable mechanical properties and slower degradation kinetics resulted in greater support of tissue remodeling in the more crosslinked scaffolds and preservation of existing myofiber area in both dECM groups compared to the empty defect control. More sustained release of pro-regenerative degradation products also promoted greater myofiber formation in the defect site. This study allowed for a greater understanding of how electrospun skeletal muscle scaffolds interact with existing skeletal muscle and can inform their potential as a therapy in a wide variety of soft tissue applications.

Machine Learning-Guided Three-Dimensional Printing of Tissue Engineering Scaffolds.

Various material compositions have been successfully used in 3D printing with promising applications as scaffolds in tissue engineering. However, identifying suitable printing conditions for new materials requires extensive experimentation in a time and resource-demanding process. This study investigates the use of Machine Learning (ML) for distinguishing between printing configurations that are likely to result in low-quality prints and printing configurations that are more promising as a first step toward the development of a recommendation system for identifying suitable printing conditions. The ML-based framework takes as input the printing conditions regarding the material composition and the printing parameters and predicts the quality of the resulting print as either "low" or "high." We investigate two ML-based approaches: a direct classification-based approach that trains a classifier to distinguish between low- and high-quality prints and an indirect approach that uses a regression ML model that approximates the values of a printing quality metric. Both modes are built upon Random Forests. We trained and evaluated the models on a dataset that was generated in a previous study, which investigated fabrication of porous polymer scaffolds by means of extrusion-based 3D printing with a full-factorial design. Our results show that both models were able to correctly label the majority of the tested configurations while a simpler linear ML model was not effective. Additionally, our analysis showed that a full factorial design for data collection can lead to redundancies in the data, in the context of ML, and we propose a more efficient data collection strategy.

Increased human AP endonuclease 1 level confers protection against the paternal age effect in mice.

Increased paternal age is associated with a greater risk of producing children with genetic disorders originating from de novo germline mutations. Mice mimic the human condition by displaying an age-associated increase in spontaneous mutant frequency in spermatogenic cells. The observed increase in mutant frequency appears to be associated with a decrease in the DNA repair protein, AP endonuclease 1 (APEX1) and Apex1 heterozygous mice display an accelerated paternal age effect as young adults. In this study, we directly tested if APEX1 over-expression in cell lines and transgenic mice could prevent increases in mutagenesis. Cell lines with ectopic expression of APEX1 had increased APEX1 activity and lower spontaneous and induced mutations in the lacI reporter gene relative to the control. Spermatogenic cells obtained from mice transgenic for human APEX1 displayed increased APEX1 activity, were protected from the age-dependent increase in spontaneous germline mutagenesis, and exhibited increased apoptosis in the spermatogonial cell population. These results directly indicate that increases in APEX1 level confer protection against the murine paternal age effect, thus highlighting the role of APEX1 in preserving reproductive health with increasing age and in protection against genotoxin-induced mutagenesis in somatic cells.

Human O6 -methylguanine-DNA methyltransferase containing C145A does not prevent hepatocellular carcinoma in C3HeB/FeJ transgenic mice.

The prevalence of hepatocellular carcinoma (HCC) was diminished from 60% to 18% at 15 months of age in C3HeB/FeJ male transgenic mice expressing hMGMT in our previous studies. To directly test if the methyltransferase activity is required for diminished tumor prevalence, two separate lines of transgenic mice bearing an enzymatically inactive form of hMGMT were used. In these lines, cysteine 145 was substituted with alanine (C145A). Expression of the hMGMT C145A transgene in liver was demonstrated by Northern blots and Western blots. Immunohistochemistry revealed predominantly nuclear localization of the hMGMT C145A protein. hMGMT C145A transgenic mice were crossed with lacI transgenic mice to assess mutant frequencies in the presence of the mutant protein. Mutant frequencies were similar among livers of lacI × hMGMT C145A bi-transgenic mice and lacI × wild-type (WT) mice. DNA sequence analysis of recovered lacI mutants revealed similar mutation spectra for hMGMT C145A and WT mice. The prevalence of HCC was also similar for the two tested lines of hMGMT C145A mice, 45% and 48% prevalence with median tumor sizes of 11 and 8 mm, and WT mice, 40% prevalence and median tumor size of 10 mm. These results provide evidence that residue C145 in hMGMT is required to reduce the prevalence of HCC in C3HeB/FeJ mice transgenic for hMGMT.

Age-related instability in spermatogenic cell nuclear and mitochondrial DNA obtained from Apex1 heterozygous mice.

The prevalence of spontaneous mutations increases with age in the male germline; consequently, older men have an increased risk of siring children with genetic disease due to de novo mutations. The lacI transgenic mouse can be used to study paternal age effects, and in this system, the prevalence of de novo mutations increases in the male germline at old ages. Mutagenesis is linked with DNA repair capacity, and base excision repair (BER), which can ameliorate spontaneous DNA damage, decreases in nuclear extracts of spermatogenic cells from old mice. Mice heterozygous for a null allele of the Apex1 gene, which encodes apurinic/apyrimidinic endonuclease I (APEN), an essential BER enzyme, display an accelerated increase in spontaneous germline mutagenesis early in life. Here, the consequences of lifelong reduction of APEN on genetic instability in the male germline were examined, for the first time, at middle and old ages. Mutant frequency increased earlier in spermatogenic cells from Apex1(+/-) mice (by 6 months of age). Nuclear DNA damage increased with age in the spermatogenic lineage for both wild-type and Apex1(+/-) mice. By old age, mutant frequencies were similar for wild-type and APEN-deficient mice. Mitochondrial genome repair also depends on APEN, and novel analysis of mitochondrial DNA (mtDNA) damage revealed an increase in the Apex1(+/-) spermatogenic cells by middle age. Thus, Apex1 heterozygosity results in accelerated damage to mtDNA and spontaneous mutagenesis, consistent with an essential role for APEN in maintaining nuclear and mtDNA integrity in spermatogenic cells throughout life.