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Although bone-patellar tendon-bone (B-PT-B) autografts are the gold standard for repair of anterior cruciate ligament ruptures, they suffer from drawbacks such as donor site morbidity and limited supply. Engineered tissues modeled after B-PT-B autografts are promising alternatives because they have the potential to regenerate connective tissue and facilitate osseointegration. Towards the long-term goal of regenerating ligaments and their bony insertions, the objective of this study was to construct 2D meshes and 3D cylindrical composite scaffolds - possessing simultaneous region-wise differences in fiber orientation, diameter, chemistry and mechanical properties - by electrospinning two different polymers from off-set spinnerets. Using a dual drum collector, 2D meshes consisting of an aligned polycaprolactone (PCL) fiber region, randomly oriented poly(lactide-co-glycolide) (PLGA) fiber region and a transition region (comprised of both PCL and PLGA fibers) were prepared, and region-wise differences were confirmed by microscopy and tensile testing. Bone marrow stromal cells (BMSCs) cultured on these meshes exhibited random orientations and low aspect ratios on the random PLGA regions, and high aspect ratios and alignment on the aligned PCL regions. Next, meshes containing an aligned PCL region flanked by two transition regions and two randomly oriented PLGA regions were prepared and processed into 3D cylindrical composite scaffolds using an interpenetrating photo-crosslinkable polyethylene glycol diacrylate hydrogel to recapitulate the shape of B-PT-B autografts. Tensile testing indicated that cylindrical composites were mechanically robust, and eventually failed due to stress concentration in the aligned PCL region. In summary, this study demonstrates a process to fabricate electrospun meshes possessing region-wise differences in properties that can elicit region-dependent cell responses, and be readily processed into scaffolds with the shape of B-PT-B autografts.

Although age-at-injury influences chronic recovery from traumatic brain injury (TBI), the differential effects of age on early outcome remain understudied. Using a male murine model of moderate contusion injury, we investigated the underlying mechanism(s) regulating the distinct response between juvenile and adult TBI. We demonstrate similar biomechanical and physical properties of naive juvenile and adult brains. However, following controlled cortical impact (CCI), juvenile mice displayed reduced cortical lesion formation, cell death, and behavioral deficits at 4 and 14 d. Analysis of high-resolution laser Doppler imaging showed a similar loss of cerebral blood flow (CBF) in the ipsilateral cortex at 3 and 24 h post-CCI, whereas juvenile mice showed enhanced subsequent restoration at 2-4 d compared with adults. These findings correlated with reduced blood-brain barrier (BBB) disruption and increased perilesional vessel density. To address whether an age-dependent endothelial cell (EC) response affects vessel stability and tissue outcome, we magnetically isolated CD31 ϩ ECs from sham and injured cortices and evaluated mRNA expression. Interestingly, we found increased transcripts for BBB stability-related genes and reduced expression of BBB-disrupting genes in juveniles compared with adults. These differences were concomitant with significant changes in miRNA-21-5p and miR-148a levels. Accompanying these findings was robust GFAP immunoreactivity, which was not resolved by day 35. Importantly, pharmacological inhibition of EC-specific Tie2 signaling abolished the juvenile protective effects. These findings shed new mechanistic light on the divergent effects that age plays on acute TBI outcome that are both spatial and temporal dependent.