Regeneration of growth plates in the long bones of the neonatal rat hindlimb

Twenty-seven male albino rats underwent hindlimb amputations through the lower femur or the midshaft of the tibiofibula on the tenth to 12th day of life. Amputation stumps were examined grossly and histologically in order to assess the significance of level and angle of transaction as determinants of subsequent growth and regeneration and to ascertain whether growth plates can regenerate following their complete excision. Amputees survived for 17-73 days. In order to exclude limbs which had been severed at or distal to the level of the growth plate, amputated limb segments either were cleared to transparency and inspected under low magnification or were sectioned serially and examined by using a compound microscope. Following amputations through the femur, the predominant response involved repair of the skeletal defect and healing of adjacent soft tissues (ten of 17 rats). Among five other animals the skeletal terminus was covered with a plate of cartilage which, in three, included areas of growth-plate architecture. Two additional transfemoral amputees regenerated incomplete growth plates, each overlying a single epicondylar surface, and one provided with a regenerated hemiepiphysis. Five of ten transtibiofibular amputees formed cartilage plates which covered the skeletal terminus in whole or in part and one regenerated an entire growth plate restricted to the distal fibula. It is concluded that angle and level of transection are not pivotal modifiers of growth and regeneration processes, and that distal growth plates may regenerate entirely or in part following their complete removal from the hindlimb.     

Hyaluronidase activity and glycosaminoglycan synthesis in the amputated newt limb: comparison of denervated, nonregenerating limbs with regenerates.

Amputated, regenerating forelimbs have been compared with the contralateral, denervated non-regenerating limb stumps in the adult newt Notophthalmus viridescens, with respect to hyaluronidase activity and the incorporation of ³H-acetate into glycosaminoglycans (GAG). At 10 days after amputation, which is the time of maximum hyaluronate production in the early growing regenerate, incorporation of ³H-acetate into GAG (cpm/mg protein) in the denervated, nonregenerating limb stump was approximately 50% of that in the contralateral regenerating limbs. At this stage, hyaluronate was the major GAG being produced, but the ratio of incorporation into hyaluronate relative to chondroitin sulfate was reduced in the denervated limbs. In intact, nonamputated limbs, the incorporation into GAG was 5% of that in the regenerating limb 10 days after amputation, and 10% of that in the denervated stumps.At 25 days, cartilage is forming and chondroitin sulfate synthesis predominates in the normal regenerate whilst the contralateral, denervated limb stumps are forming scars. GAG synthesis in the latter was less than one-quarter the level seen in the regenerating limbs, mostly due to low incorporation into chondroitin sulfate.Hyaluronidase activity, which appears in the regenerating limb during differentiation of skeletal elements (20–45 days), was not detectable in limbs denervated early enough to prevent regeneration. However, limbs denervated after formation of the blastema will regenerate without nerve, and hyaluronidase activity in such limbs was normal. Thus, hyaluronidase activity appears when regeneration reaches the cartilage deposition stage, with or without nerve.     

Forelimb regeneration in the postmetamorphic bullfrog: stimulation by dimethyl sulfoxide and retinoic acid

To examine the effects of retinoic acid and dimethyl sulfoxide on regenerative ability of anuran amphibians, the left forelimbs of 60 postmetamorphic froglets of Rana catesbeiana (bullfrogs) were amputated through the distal zeugopodium. Fifteen of the froglets had their left forelimb stumps immersed in dimethyl sulfoxide (DMSO) for 3 minutes, once immediately after amputation and once on each of 5 subsequent days. Another 15 frogs had their left forelimb stumps immersed in a 0.01 M solution of retinoic acid dissolved in DMSO for the same period of time. The remaining 30 control froglets did not regenerate structures distal to the amputation surface, while all limbs in both treated groups produced regenerates by 120 days postamputation. Regenerates of limbs treated with both DMSO alone and DMSO combined with retinoic acid, although hypomorphic, were composed of multiple cartilage elements, which in many cases (46.7%) were organized as patterns partially resembling the skeletal arrangement of a normal forelimb. All of these regenerates exhibited bundles of striated muscle. In addition, nearly half (46.7%) of the regenerates in the DMSO + retinoic acid group possessed two separate regenerate outgrowths. The results demonstrate that young bullfrogs (Rana catesbeiana) possess a latent epimorphic regenerative capability, which can be stimulated by topical application to the wound surface of DMSO alone or DMSO combined with retinoic acid.     

Regeneration of appendages in the large milkweed bug, Oncopeltus fasciatus

An analysis was made of the regeneration of legs and antennae of Oncopeltus. Amputations were performed on first instar larvae within 24 hr after hatching, and on later instars within 24 hr after ecdysis. The resulting regenerates were then measured at each instar. When amputations were performed soon after hatching, there was no significant effect on the duration of any instar. The regenerate was usually visible after the second post-operative ecdysis, and was smaller than a normal appendage (hypomorphic). Removal of the three distal segments of the antenna usually resulted in regeneration of only one segment which was abnormally long and showed a combination of the bristle patterns characteristic of the two most distal segments of the control. In a few such cases a partial intersegmental membrane was present in the regenerated segment. Removal of the tarsus resulted in a structurally complete regenerate which was smaller than the control tarsus. The largest leg regenerates were obtained when amputation was performed through the tibia. With amputation through the femur, a decrease in length of the remainder of this segment was observed after the first ecdysis. This type of amputation and amputation through the trochanter in some cases resulted in the formation of a globular stump containing tarsal claws. The results indicate that amputation of part of an appendage in Oncopeltus does not stimulate an increased growth rate in the stump, but merely causes reorganization of the stump material which subsequently grows at the normal rate. Since even the most hypomorphic regenerates contained well-formed claws, even though proximal parts were missing, it appears that the reorganization process must begin at the most distal point and proceed proximally.     

Supernumerary regeneration in the large milkweed bug Oncopeltus fasciatus.

As an approach to the problem of pattern formation in the insect appendage, various graft combinations were studied in the legs of the large milkweed bug Oncopeltus fasciatus. Metathoracic legs of fourth instar larvae were amputated through the tibia within 24 hr after ecdysis and grafted back onto the stumps. The orientation of the graft was altered by rotation through 90 or 180° and/or by exchanging right and left stumps and grafts, yielding seven possible orientations in addition to the control. Many of these grafts resulted in the production of one or two supernumerary regenerates of the distal segments, which appeared at the graft junction after the second postoperative ecdysis. When two supernumerary regenerates resulted, one appeared to be produced from the stump and the other from the graft. When one regenerate was present, it appeared to be a composite of material produced from both the stump and the graft. In contrast to the results obtained in cockroaches, the external face of the leg appeared to be the only one capable of giving rise to a supernumerary regenerate.     

Antler regeneration: a dependent process of stem tissue primed via interaction with its enveloping skin

Deer antlers are unique mammalian appendages in that each year they are cast and fully regenerate from permanent bony protuberances, called pedicles. In a previous study, we found that there is a difference in the degree of association between pedicle bone and its enveloping skin: tight at the distal third and loose at the proximal two thirds of a pedicle stump. The distal part has been termed the "potentiated" region, and the proximal part the "dormant" region. In the present study, pedicle stumps were artificially created in yearling sika deer by cutting off the tissue distal to either the potentiated or the dormant region. A piece of impermeable membrane was then inserted into the space between the bone and the skin of each treated pedicle stump, while the control pedicles had the same surgery without membrane insertion. The results showed that the inserted membrane blocked pedicle skin participation in the process of antler regeneration. All three potentiated bony pedicle stumps regenerated skin-less antlers; whereas, one of the three dormant bony pedicle stumps failed to regenerate any antler tissue. The other two dormant stumps eventually regenerated normal antlers; however, this only occurred after loss of the inserted membrane. No antler tissue regenerated from the dormant stumps while the inserted membrane remained in place (up to 55 days). All control pedicle stumps regenerated normal antlers. Therefore, we conclude that it is the pedicle bone, but not pedicle skin, that gives rise to regenerating antlers, and that pedicle bone can acquire the potential to regenerate an antler only when it is primed via interaction with its enveloping skin.     

Tissue regeneration during lower jaw restoration in zebrafish shows some features of epimorphic regeneration

Zebrafish have the ability to regenerate skeletal structures, including the fin, skull roof, and jaw. Although fin regeneration proceeds by epimorphic regeneration, it remains unclear whether this process is involved in other skeletal regeneration in zebrafish. Initially in epimorphic regeneration, the wound epidermis covers the wound surface. Subsequently, the blastema, an undifferentiated mesenchymal mass, forms beneath the epidermis. In the present study, we re-examined the regeneration of the zebrafish lower jaw in detail, and investigated whether epimorphic regeneration is involved in this process. We performed amputation of the lower jaw at two different positions; the proximal level (presence of Meckel's cartilage) and the distal level (absence of Meckel's cartilage). In both manipulations, a blastema-like cellular mass was initially formed. Subsequently, cartilaginous aggregates were formed in this mass. In the proximal amputation, the cartilaginous aggregates were then fused with Meckel's cartilage and remained as a skeletal component of the regenerated jaw, whereas in the distal amputation, the cartilaginous aggregates disappeared as regeneration progressed. Two molecules that were observed during epimorphic regeneration, Laminin and msxb, were expressed in the regenerating lower jaw, although the domain of msxb expression was out of the main plain of the aggregate formation. Administration of an inhibitor of Wnt/β-catenin signaling, a pathway associated with epimorphic regeneration, showed few effects on lower jaw regeneration. Our finding suggests that skeletal regeneration of the lower jaw mainly progresses through tissue regeneration that is dependent on the position in the jaw, and epimorphic regeneration plays an adjunctive role in this regeneration.     

Analytical study of Xenopus hindlimb regenerate with special reference to muscle regeneration

Amputated hindlimbs of Xenopus laevis, develop various types of regenerates in relation with amputation level as well as stage development. The present experiments is an attempt to study the histological characteristics of Xenopus regenerations, i.e., rational changes of tissue components along the length of the regenerated part with special emphasis on the degree of muscle regeneration. Four types of regenerates were studied viz; a 4th toe obtained from a completely restored regenerated limb at 126 days after amputation of limb at base level in stage 51. An amputated limb with no external sign of regeneration of limb at thigh level in stage 60. A spike-shaped regenerate at 96 days after amputation of limb at shank level in stage 63. A spike-shaped regenerate at about 2 years after amputation of limb at shank level in stage 60. Cross sectional areas of muscle, skin gland, epidermis and cartilage in each of the four types of regenerates were measured with Image Analyzing Apparatus (VIP 121 CH, Olympus Co.). The relative area of each tissue was expressed as a percentage of the cross sectional area of the limb. The obtained values were plotted along the length of the regenerate. Digitiform regenerates were found to be more or less similar to the control limbs, i.e., provided joints and muscle, while the heteromorphic spike or rod shaped regenerates were simply provided with cartilaginous axial core without joint formation. Muscle area were reduced rapidly near the amputation area of these heteromorphic regenerates with no more continuation in the regenerated tissue. It is interesting to mention that percentage cartilage area of about 2 years old spike regenerate was higher than that of similar 96 days regenerate. In addition muscle regeneration was completely absent even in such an aged regenerate. The area showed fairly similar ratio irrespective of the external appearance of the regenerate. In 32 regenerates of which limbs were amputated at various developmental stages ranging between stage 51 and adult stage, the histological condition of muscle at the amputation site, were well observed. In all digitated types of regenerates even in those with reduced number of toes, muscles were found grown well in the regenerates. In heteromorphic regenerates without toe formation muscle did not usually regenerate. In few cases, however, a small mass of myoblastic like cells or small aggregation of differentiated muscle cells without any structural continuation with the stump muscles, were seen to develop in the midst of the regenerate.(ABSTRACT TRUNCATED AT 400 WORDS)     

Transient inability of neonatal rat motoneurons to reinnervate muscle

We studied the reinnervation of internal intercostal muscles of newborn rats. The distal halves were denervated by nerve section at various ages between birth and 6 weeks. Regardless of the age at denervation, neither evoked nor spontaneous nerve-muscle transmission reappeared until the animals were at least 3 weeks old. Older rats recovered a substantial degree of function within 7 days of nerve section. Normally the motor units in this muscle are narrowly distributed, so most axotomized motoneurons lost their entire synaptic periphery. Reinnervation was by axons which had been sectioned, and regenerated motor units were of normal size and number. There was no collateral sprouting from end plates left intact. Motoneurons axotomized at birth did regenerate axons the full length of the muscle within 7 days of operation. Their failure to reinnervate the muscle was due to delay in forming functional end plates. Nerve section in animals aged 1 month or older resulted in an abnormal pattern of reinnervation; reinnervated motor units were diffusely spread through large portions of the muscle, although they still did not overlap with the region left intact. This indicates that thoracic motoneurons respond to axotomy differently in neonatal rats than they do in adults.     

肢体再生——来自有尾两栖类的认知

蝾螈等有尾两栖类在其肢体任何节段被截断后,能通过准确的时空模式调节完成具有位置匹配关系的再生修复,该过程由受损肢体残端产生的芽基组织介导完成。芽基细胞的来源目前尚有争议,其产生受局部基质微环境诱导并涉及细胞表观遗传学改变,性状上呈现不完全的细胞再编程特征,增殖分化具有神经依赖性。哺乳类包括人类仅具有极为有限的肢体再生能力,其肢体再生限于指（趾）末端受损离断。深入探讨有尾两栖类等肢体再生过程的细胞分子机制,将为探索新的干细胞损伤修复途径及再生促进策略提供线索。     

Influence of clenbuterol on bone metabolism in exercised or sedentary rats.

This paper reports that the selective beta(2)-adrenergic receptor agonist clenbuterol affects bone metabolism in growing 3-mo-old male Wistar rats treated over 8 wk. Thirty-two 3-mo-old growing Wistar rats weighing 234 +/- 2 g were assigned to a progressive isometric force, strength-training exercise program plus oral clenbuterol (2 mg x kg body wt(-1) x day(-1)) for 5 days each week, exercise program without clenbuterol 5 days each week, no exercise program plus oral clenbuterol (2 mg x kg(-1) x day(-1)) for 5 days each week, or no exercise without clenbuterol 5 days each week. At the end of 8 wk, lean mass, fat mass, and right total femoral, distal metaphyseal femoral, and diaphyseal femoral bone mineral density were measured by Hologic QDR 4,500 dual X-ray absorptiometry (DEXA) technique. Left femoral bones were harvested after death on day 58, and femoral resistance was determined by three-point bending testing. We found that fat mass was decreased in rats given strength training exercise and decreased further in rats treated with clenbuterol. Lean mass was increased in clenbuterol-treated animals. Strength-training exercise appeared to have no effect on bone mineral density, serum osteocalcin, or urinary deoxypyridinoline. However, clenbuterol treatment decreased femoral length, diameter, bone mineral density, and mechanical resistance. Clenbuterol had no effect on osteocalcin but increased urinary deoxypyridinoline. We concluded that clenbuterol treatment decreased bone mineral density and increased bone resorption independent of the level of exercise rats were given.     

Electric currents in Xenopus tadpole tail regeneration

Xenopus laevis tadpoles can regenerate tail, including spinal cord, after partial amputation, but lose this ability during a specific period around stage 45. They regain this ability after stage 45. What happens during this “refractory period” might hold the key to spinal cord regeneration. We hypothesize that electric currents at amputated stumps play significant roles in tail regeneration. We measured electric current at tail stumps following amputation at different developmental stages. Amputation induced large outward currents leaving the stump. In regenerating stumps of stage 40 tadpoles, a remarkable reversal of the current direction occurred around 12-24 h post-amputation, while non-regenerating stumps of stage 45 tadpole maintained outward currents. This reversal of electric current at tail stumps correlates with whether tails regenerate or not (regenerating stage 40—inward current; non-regenerating stage 45—outward current). Reduction of tail stump current using sodium-free solution decreased the rate of regeneration and percentage regeneration. Fin punch wounds healed normally at stages 45 and 48, and in sodium-free solution, suggesting that the absence of tail re-growth at stage 45 is regeneration-specific rather than a general inhibition of wound healing. These data suggest that electric signals might be one of the key players regulating regeneration.     

Development and regeneration of the neonatal digit tip in mice

The digit tips of children and rodents are known to regenerate following amputation. The skeletal structure that regenerates is the distal region of the terminal phalangeal bone that is associated with the nail organ. The terminal phalanx forms late in gestation by endochondral ossification and continues to elongate until sexual maturity (8 weeks of age). Postnatal elongation at its distal end occurs by appositional ossification, i.e. direct ossification on the surface of the terminal phalanx, whereas proximal elongation results from an endochondral growth plate. Amputation through the middle of the terminal phalanx regenerates whereas regenerative failure is observed following amputation to remove the distal 2/3 of the bone. Regeneration is characterized by the formation of a blastema of proliferating cells that appear undifferentiated and express Bmp4. Using chondrogenic and osteogenic markers we show that redifferentiation does not occur by endochondral ossification but by the direct ossification of blastema cells that form the rudiment of the digit tip. Once formed the rudiment elongates by appositional ossification in parallel with unamputated control digits. Regenerated digits are consistently shorter than unamputated control digits. Finally, we present a case study of a child who suffered an amputation injury at a proximal level of the terminal phalanx, but failed to regenerate despite conservative treatment and the presence of the nail organ. These clinical and experimental findings expand on previously published observations and initiate a molecular assessment of a mammalian regeneration model.     

Morphogenetic control during tail regeneration in Plethodon cinereus: the role of skeletal muscle

The caudal myofibers of Plethodon cinereus do not appear to participate directly in epimorphic tail regeneration following either autotomy or surgical amputation of the tail. The possibility that tail musculature might indirectly influence morphogenesis of the regenerate was tested by unilaterally removing 99% of the lateral muscle mass for five to six caudal segments. Ten days after muscle ablation, tails were amputated through the deficient area. Unlike previous experiences with ambystomid larvae, P. cinereus regulates completely producing a normal tail regenerate and at a rate comparable to that following simple amputation.     

Regenerative responses in cultured hindlimb stumps of larval Xenopus laevis.

The regenerative capacity of larval Xenopus laevis hindlimbs amputated through the tarsalia at different stages of development and explanted in vitro was tested. In the first experimental series hindlimb stumps from stage 53, 54, 55, and 57 larvae (according to Nieuwkoop and Faber, '56) were cultured in Leibovitz's L-15 medium supplemented with 10% FCS, and 0.04 U of insulin and 10(-8) mg of L-thyroxine per ml of medium. Results showed that the distal part of the limb stumps from stages 53, 54, and 55 formed a regeneration blastema composed of proliferating mesenchymal cells beneath a typical apical cap. No blastema was formed in the proximal part of the stump. In limb stumps from stage 57, a regeneration blastema did not form either in the proximal or in the distal part of the stump. In a second experimental series, hindlimb stumps from stage 55 larvae, denervated 5 days prior to amputation in order to eliminate any residual neurotrophic factor, were cultured in a simplified L-15 medium containing 2% FCS and lacking insulin and thyroxine. Results showed that also in these experimental conditions the stumps from stage 55 formed a conical regeneration blastema at the distal tip. The blastema cells duplicated their own DNA and divided. At the proximal extremity no regeneration blastema was formed. In the same culture medium, the stumps of larvae at stage 57 did not form a regeneration blastema.(ABSTRACT TRUNCATED AT 250 WORDS)     

Experimental regeneration of the caudal skeleton of the glass knifefish,Eigenmannia virescens (Rhamphichthydae,Gymnotoidei)

This paper describes the regeneration of the caudal axial skeleton after amputation of the tail, including about 20 vertebrae, in the gymnotoid fish Eigenmannia virescens. Seven days after amputation, a regeneration blastema developed and soft tissues degenerated. A cylinder of cartilage developed at the end of the notochord. When this cartilage was about 10 mm long (21 days), perichondral ossification began. The cartilage continued to elongate and ossification increased while osteoclasts began to destroy the cartilage ventrally. Finally, a bony rod formed and at its tip the cartilage persisted as a rod, 2 to 3 mm long. The anal fin also regenerated: Endoskeletal cartilage developed first, following by differentiation of the lepidotrichia, and finally ossification of the endoskeleton.     

Regeneration of whole limbs from shank stumps in toad tadpoles treated with vitamin A

Summary Young tadpoles of the toad,Bufo melanostictus (Schneider), were immersed in 15 IU/ml vitamin-A palmitate solution for 3 days, only prior to amputation through the shank. In more than 65% of cases the resultant regenerates were whole limbs containing the skeletal elements from femur to phallanges; in several of them a new girdle had also differentiated. In others regenration had progressed only up to the blastema stage and postblastemic development was inhibited. Opposite results were obtained when treatment was extended to another 3 days after amputation. A normal control-type regenerate consisting of parts distal to amputation level was not obtained in any case treated in either manner. The removed distal part of the shank was not restored in any treated case. It appears that, if suitably administered, vitamin A can make the limb regeneration blastema of amphibians completely equivalent to the original limb bud, probably by intensifying dedifferentiation of its cells. It is suggested that this chemical can be a useful tool to investigate the biochemical and genetic changes which occur during dedifferentiation and also whether through this process differentiated cells can really revert to a pluripotent state.     

The irradiated epidermis inhibits newt limb regeneration by preventing blastema growth. A histological study

It has been established that X-ray irradiation localized to a forelimb or entire irradiation of premetamorphic Pleurodeles larvae prevented limb regeneration. Transplantation of non-irradiated skin, dermis or muscle to limb stumps of locally irradiated newts was sufficient to allow a blastema to develop. Transplantation of the same tissues to limb stumps of entirely irradiated newts yielded different results with the different graft types. Skin graft allowed a normal blastema to be established but dermis or muscle grafts did not. In order to define more precisely the role played by the epidermis in the establishment of a blastema, and in the growth of a regenerate, different combinations of limb tissues, either irradiated or not, were carried out at the level of amputated limb stumps. At four different times (8-10 days; 13-15 days; 20-23 days; 30 days or more) after amputation the stumps were examined in histological longitudinal sections to study the first events of regeneration, that is dedifferentiation and growth. Dedifferentiation occurred in both normal and irradiated tissues of mesodermal origin. The healthy mesenchymal cells began dividing and formed a growing blastema only when associated with a non-irradiated epidermis. Healthy mesenchymal cells covered with an irradiated epidermis exhibited a few mitoses after dedifferentiation, but the mitotic figures became rarer and rarer until the animals died. The lack of dense accumulation of blastemal cells in such limb stumps suggested that the healthy epidermis allows the mesenchymal cells to divide actively to constitute a growing blastema. Hence, X-ray irradiation seems to be responsible for the loss of such an epidermal mitogenic influence on the underlying mesenchymal cells.     

Ultrastructural features of the process of wound healing after tail and limb amputation in lizard

Alibardi, L. 2010. Ultrastructural features of the process of wound healing after tail and limb amputation in lizard.—Acta Zoologica (Stockholm)  91 : 306–318 Wound healing and re‐epitelization after amputation of tail and limb in lizard have been studied by electron microscopy to understand the cytological base of immunity to infection in this species. After 2 days post‐amputation in both limb and tail stumps, numerous granulocytes are accumulated over the stump, and participate to the formation of the scab. Bacteria remain confined to the scab or are engulfed by leukocytes and migrating keratinocytes located underneath the scab. Bacteria are degraded within lysosomes present in these cells and are not observed among mesenchymal cells or in blood vessels of the regenerative blastema. Granulocytes, migrating keratinocytes, and later macrophages form an effective barrier responsible for limiting microbe penetration. The innate immunity in lizard is very effective in natural (dirty) condition and impedes the spreading of infection to inner tissues. While the complete re‐epitelization of the tail stump underneath the scab requires 4–7 days, the same process in the limb requires 8–18 or more days post‐amputation, depending from the level of amputation and the persistence of a protruding humerus or femurs on the stump surface. This delay produces the permanence of inflammatory cells such as granulocytes and macrophages in the limb stump for a much longer period than in the tail stump, a process that stimulates scarring.     

Normal newt limb regeneration requires matrix metalloproteinase function

Newts regenerate lost limbs through a complex process involving dedifferentiation, migration, proliferation, and redifferentiation of cells proximal to the amputation plane. To identify the genes controlling these cellular events, we performed a differential display analysis between regenerating and nonregenerating limbs from the newt Notophthalmus viridescens. This analysis, coupled with a direct cloning approach, identified a previously unknown Notophthalmus collagenase gene (nCol) and three known matrix metalloproteinase (MMP) genes, MMP3/10a, MMP3/10b, and MMP9, all of which are upregulated within hours of limb amputation. MMP3/10b exhibits the highest and most ubiquitous expression and appears to account for the majority of the proteolytic activity in the limb as measured by gel zymography. By testing purified recombinant MMP proteins against potential substrates, we show that nCol is a true collagenase, MMP9 is a gelatinase, MMP3/10a is a stromelysin, and MMP3/10b has an unusually broad substrate profile, acting both as a stromelysin and noncanonical collagenase. Exposure of regenerating limbs to the synthetic MMP inhibitor GM6001 produces either dwarfed, malformed limb regenerates or limb stumps with distal scars. These data suggest that MMPs are required for normal newt limb regeneration and that MMPs function, in part, to prevent scar formation during the regenerative process.