Daniel A. Neufeld

Bone regrowth after digit tip amputation in mice is equivalent in adults and neonates.

Despite clinical and experimental reports of digit tip regrowth, bone regrowth after amputation through terminal phalanges has not been methodically documented. We have examined bone regrowth in mice after amputation through the terminal phalanx to determine how the level of amputation affects the response and whether the response varies between adults and neonates. Digit tips were amputated, and, at selected intervals greater than 5 weeks after amputation, digits were photographed and processed for whole mount staining with alizarin red stain. Amputations within the distal 40% of the terminal phalanx yielded grossly normal digits within 5 weeks and bone regrowth which usually attained or surpassed the original length. Amputations through the proximal 20% of the distal phalanx precluded nail plate regrowth, and bone grew minimally or regressed. Amputation through the intermediate 40% of the distal phalanx produced digit tips in which bone regrowth was correlated with nail regrowth and in which bone regrowth did not reach original levels. The response in adults and neonates was similar. The correlation between nail regrowth and bone regrowth may help to predict phalangeal bone regrowth after amputation in adults and juveniles.

Electrospun Nanofibrous Polycaprolactone Scaffolds for Tissue Engineering of Annulus Fibrosus

The annulus fibrosus comprises concentric lamellae that can be damaged due to intervertebral disc degeneration; to provide permanent repair of these acquired structural defects, one solution is to fabricate scaffolds that are designed to support the growth of annulus fibrosus cells. In this study, electrospun nanofibrous scaffolds of polycaprolactone are fabricated in random, aligned, and round-end configurations. Primary porcine annulus fibrosus cells are grown on the scaffolds and evaluated for attachment, proliferation, and production of extracellular matrix. The scaffold consisting of round-end nanofibers substantially outperforms the random and aligned scaffolds on cell adhesion; additionally, the scaffold with aligned nanofibers strongly affects the orientation of cells.

Tissue engineering of annulus fibrosus using electrospun fibrous scaffolds with aligned polycaprolactone fibers

In tissue engineering, it is important to fabricate a three-dimensional scaffold that resemble the extracellular matrix (ECM) and topographical appearance of native tissue. The aim of this study is to test the hypothesis that varying microstructures of electrospun fibrous scaffolds by manipulating the relative degree of fiber alignment would influence the behaviors of porcine annulus fibrosus cells. Five types of electrospun fibrous scaffolds with polycaprolactone fibers having random or partially aligned arrangements have been prepared and investigated. The scaffold microstructures have been examined, and in vitro experiments have been carried out to assess cell-material interaction, cell proliferation, and ECM production. The results indicate that the scaffold with oriented fibers provides strong guidance to the cell orientation and ECM distribution. In addition, albeit the tensile moduli of electrospun fibrous scaffolds are lower than that of native tissue, they are comparable to those reported in literature; hence, the constructs cultured with optimized conditions including the scaffold material selection and dynamic mechanical conditioning would have the potential to possess the moduli closer to that of native tissue.

Perspective: A suggested role for basement membrane structures during newt limb regeneration

Interactions between epithelium and mesenchyme, which occur across a basement membrane (BM) zone, are essential to generate a growth bud, or blastema, from which a new limb regenerates. An intact BM at that interface is believed to inhibit regeneration, but that mechanism of inhibition is not understood.

Bone growth is induced by nail transplantation in amputated proximal phalanges.

Abstract. Mammals are able to regrow the tips of amputated fingers and toes. However, regrowth is limited to regions covered by, and is dependent upon, the presence of the nail organ. If the nail organ is responsible for bone growth in digit-tips, we reasoned that transplanted nail organ might also be able to induce outgrowth from other levels of the digit. Partial nail organ has been transplanted to amputated proximal phalanges of young rats. To date, six transplants have successfully produced outgrowth of nail. New bone growth, not seen in control amputated digits, was documented by x-ray and by alizarin red and calcein injections to be directed toward implanted nail organ. These results support an inductive role for nail organ epithelium in bone growth after amputation and provide encouragement for attempts to enhance a positive outcome after appendage amputation.

Stabilizing role of the basement membrane and dermal fibers during newt limb regeneration

Following amputation of a newt limb, tissues at the amputation site undergo histolysis to give rise to a growth bud, or blastema, but they also provide a base on which the regenerate is constructed. Studies suggest that dermal tissues may differentially resist histolysis.

Persistent left superior vena cava and associated structural and functional considerations.

Potentially significant associations are presented between anomalous sys temic venous return (including both left superior vena cava and left hepatic venous drainage to the coronary sinus), a history of atrial fibrillation and a forme fruste of cor triatriatum in an elderly woman. Lack of associated structural defects or functional deficits makes it difficult to assess the frequency of occurrence in the general population of bilateral superior venae cavae in association with a persistent left hepatic vein draining into the coronary sinus. However, the potential for these systemic venous anomalies needs to be considered when unexplained arrhythmias are encountered. More specifically, the size of the coronary sinus needs to be assessed in patients with arrhythmia.

Denervation retards but does not prevent toetip regeneration

Toetips of mammals regrow after amputation by a process similar, but not identical, to that which occurs during regeneration of a newt limb. Nerve is needed as a mitotic stimulant for newt limb regeneration but the requirement for nerve during rodent digit‐tip regeneration is not known. Nerve dependence in rats was tested by severing the sciatic nerve in one hindlimb, amputating digit‐tips from the central digits of both hind feet, and comparing the amount of regrowth in innervated and denervated digits. Denervation delayed soft‐tissue wound healing. However, denervation did not significantly affect bone regrowth when animals were examined at one month. Because we suspected delayed bone regrowth, we used a new method that we developed to follow bone growth at several time points in each animal. Termed visible bone fluorescence through nail, this technique used serial injections of fluorescent calcium‐deposition markers and observation through the toenails to observe bone growth in living animals. Using this method it was possible to detect retarded bone regrowth in denervated digits. Thus, although denervation of rodent tips delayed both soft tissue healing and bone regrowth, it did not prevent ultimate restitution of the amputated part. This suggests that neurotrophic stimulation in the mammalian digit‐tip is not identical to that documented during newt limb regeneration, and that growth stimulation may be provided by tissues other than nerve.

Formation of prechondrogenic mesenchymal cell aggregates in the regenerating newt limb

Abstract Development of the pattern of prechondrogenic condensations was documented by quantitating regional cell densities within blastemas of regenerating forelimbs of newts. The pattern of development resembles that of embryonic limb development in that the prechondrogenic pattern develops from proximal to distal, and by the presence of an apical epithelial thickening which is most prominent postaxially during the intermediate phase of mesenchymal cell accumulation. Development differs from that described previously in that an increased central cell density is present in the smallest blastemas analyzed, and the sequence of digit formation is from anterior to posterior. Also, a blister often intervenes between the epithelium and mesenchyme during the early phase of development. Cumulatively, these observations support models which suggest mechanisms for proximodistal development and a stimulatory role for apical epithelium. However, the presence of a central cell density prior to definitive aggregation implies a mechanism other than de novo condensation which is assumed in current models. Additionally, increased mesenchymal cell density subjacent to thickened epithelium is contrary to models which imply that such areas should be less dense. Finally, digital aggregates develop from preaxial to postaxial, which is opposite that of most amniotes, and is not predicted by current models of limb morphogenesis.

Use of Enzyme Overlay Membranes to Survey Proteinase Activity in Frozen Sections: Cathepsin-like and Plasmin-like Activity in Regenerating Newt Limbs

We present a method that permits extremely simple and rapid screening of proteolytic enzyme activity in sectioned tissues. Enzyme overlay membranes (EOMs) are custom-made membranes designed to fluoresce at sites of specific proteolytic enzyme activity after separation of proteins by gel electrophoresis. EOMs, selected to detect either plasmin-like or cathepsin B-like activity, have been used in a novel way to document the distribution of enzyme activity in frozen sectioned tissues. When moistened membranes were placed in contact with sectioned regenerating newt limbs, a fluorescent pattern of enzyme activity was generated. In limbs at 3 hr post amputation, cathepsin B-like activity was prominent across the amputation site but plasmin-like activity was distributed in dermal and deeper proximal tissues, suggesting different roles for these two classes of enzymes. EOM enzymology in situ (EEI) on frozen sectioned tissues may be a widely useful technique to display distribution and level of activity of proteolytic enzymes in various systems.