Current tissue engineering approaches for tendon defects require improved biomaterials to balance microstructural and mechanical design criteria. Collagen-glycosaminoglycan (CG) scaffolds have shown considerable success as in vivo regenerative templates and in vitro constructs to study cell behavior. While these scaffolds possess many advantageous qualities, their mechanical properties are typically orders of magnitude lower than orthopedic tissues such as tendon. Taking inspiration from mechanically efficient core–shell composites in nature such as plant stems and porcupine quills, we have created core–shell CG composites that display high bioactivity and improved mechanical integrity. These composites feature integration of a low density, anisotropic CG scaffold core with a high density, CG membrane shell. CG membranes were fabricated via an evaporative process that allowed separate tuning of membrane thickness and elastic moduli and were found to be isotropic in-plane. The membranes were then integrated with an anisotropic CG scaffold core via freeze-drying and subsequent crosslinking. Increasing the relative thickness of the CG membrane shell was shown to increase composite tensile elastic modulus by as much as a factor of 36 in a manner consistent with predictions from layered composites theory. CG scaffold-membrane composites were found to support tendon cell viability, proliferation, and metabolic activity in vitro, suggesting they maintain sufficient permeability while demonstrating improved mechanical strength. This work suggests an effective, biomimetic approach for balancing strength and bioactivity requirements of porous scaffolds for tissue engineering.

Biomaterials for orthopedic tissue engineering must balance mechanical and bioactivity concerns. This work describes the fabrication of a homologous series of anisotropic collagen–GAG (CG) scaffolds with aligned tracks of ellipsoidal pores but increasing relative densities (ρ*/ρs), and we report the role scaffold relative density plays in directing tenocyte bioactivity. Scaffold permeability and mechanical properties, both in tension and compression, were significantly influenced by relative density in a manner predicted by cellular solids models. Equine tenocytes showed greater levels of attachment, metabolic activity, soluble collagen synthesis, and alignment as well as less cell-mediated scaffold contraction in anisotropic CG scaffolds of increasing relative density. Notably, the lowest density scaffolds experienced significant cell-mediated contraction with associated decreases in tenocyte number as well as loss of microstructural integrity, aligned contact guidance cues, and preferential tenocyte orientation over a 14 day culture period. Gene expression analyses suggested tenocyte de-differentiation in the lowest density scaffold while indicating that the highest density scaffold supported significant increases in COMP (4-fold), tenascin-C (3-fold), and scleraxis (15-fold) expression as well as significant decreases in MMP-1 (9-fold) and MMP-13 (13-fold) expression on day 14. These results suggest that anisotropic scaffold relative density can help to modulate the maintenance of a more tendon-like microenvironment and aid long-term tenocyte transcriptomic stability. Overall, this work demonstrates that relative density is a critical scaffold parameter, not only for insuring mechanical competence, but also for directing cell transcriptomic stability and behavior.

In the hydropenic intact animal, knowledge of solute and water transfers in the renal concentrating operation may be obtained by utilizing osmotic diuresis (2-5). Changes in the urine to plasma osmotic ratio (Uosm/Poum) and tubular reabsorption of solute-free water (TcH2o) with increasing rates of total . solute clearance (Co.m) and urine flow (V) may give information pertaining to the limits of the mechanisms involved in the concentration of urine. Much of the data obtained from normal man with this technique has been derived from studies employing hypertonic mannitol as the loading solute (3, 5). These studies have characteristically shown that, during mannitol diuresis in hydropenia, TCH2O achieves an upper limit or maximum when Co.m exceeds 15 ml per minute and that urine osmolality approaches the osmolality of plasma as a limit. If TCH2O is an index of water movement from the collecting duct into the hypertonic medullary interstitium, then it is probably a reflection of the rate of transport and accumulation of solute (chiefly sodium chloride) in the renal medulla. The observed upper limit to TCH2O with mannitol diuresis might indicate an intrinsic maximal capacity for the active transport of sodium in the ascending limb of the loop of Henle (5, 6). This interpretation, however, is at variance with the observations made in micropuncture studies in rats during saline loading that fail to show any

Tendons are connective tissues that transmit loads between bone and muscle. The biological solution to the problem of connecting relatively compliant tendon to stiffer (∼2 orders of magnitude) bone is a gradient interface zone ∼100μm wide. Over the tendon-bone-junction (TBJ) a linear transition takes place in the ECM inorganic:organic (mineral:collagen) ratio as well as mineral crystallinity from that of tendon to bone. While small TBJ injuries can heal via regeneration, severe defects undergo repair-mediated healing characterized by fibrocartilagenous scar tissue with inferior biomechanical and functional properties. Severe TBJ injuries are common in athletes, the elderly, and following severe craniofacial and extremity trauma. Many tendon injuries (i.e. supraspinatus injuries), particularly those associated with acute trauma, are prone to occur at the TBJ due to high levels of region-specific stress concentrations; rotator cuff tendons injuries, one of the most common TBJ injuries, exhibit re-tears at rates as high as 94%. The scale of such defects and current poor clinical results suggest the need for a biomaterial solution that can mimic the dynamic heterogeneities of the native insertion and tendon body to induce rapid, functional regeneration. Three-dimensional collagen-GAG (CG) scaffolds have been successfully used clinically to regenerate large soft tissue defects (skin, peripheral nerves); they act by mimicking the native extracellular matrix (ECM) of the damaged tissue to prevent wound contraction and scar tissue synthesis. However these scaffolds have not traditionally been used for orthopedics due to an inability to recapitulate two critical features of orthopedic tissues: multiscale structural complexity, biomechanical properties.

Summary. To study the renal medullary transport and accumulation of urea in dogs independent of water transport, we obliterated the medullary electrolyte gradient by a sustained ethacrynic acid diuresis. Infusions of urea were also given at various rates to vary urinary urea concentration. In the steady state, the kidneys were removed, and slices were analyzed for water, urea, and electrolytes. In every experiment in 15 dogs over a range of urinary urea concentration from 19 to 230 mmoles per L and urine flow from 0.5 to 9.7 ml per minute per kidney, an intrarenal urea gradient persisted, and urinary urea concentration was always lower than papillary water urea concentration. The magnitude of this uphill urinary-papillary gradient (mean + SE = -21 + 2.9 mmoles per L) was not affected by hemorrhagic hypotension or a nonprotein diet.In 12 additional experiments begun similarly, inhibitors were infused into one renal artery. Both iodoacetate, an inhibitor of anaerobic glycolysis, and acetamide, an analogue of urea, markedly and significantly reduced both the intrarenal urea gradient and the uphill urinary-papillary gradient. In contrast, cyanide, an inhibitor of oxidative metabolism, had no observable effect on the urea gradients. The data are best explained by postulating an active transport system for urea in the medullary collecting duct deriving its energy from anaerobic glycolysis.