Jennifer J. Marler

TRANSPLANTATION OF CELLS IN MATRICES FOR TISSUE REGENERATION

Tissue engineering is a field that has truly emerged in the last decade. It has brought together diverse technologies, e.g. cell culture, polymer chemistry and transplantation. The creation of matrices to guide tissue regeneration allows manipulation at several levels, i.e. the cells employed, the choice of polymer and the design of construct assembly methods. We present experience using such constructs to guide regeneration of diverse tissues, e.g. liver, intestine, urologic tissue, skin, cartilage, bone and cardiovascular structures. Emerging concepts in using cell/polymer constructs include the need for appropriate modeling of the micromechanical environments of different tissues, as well as the necessity of finding new strategies to achieve vascularization of tissues for transplant. Finally, the concept of applying tissue-engineered structures to non-native sites is discussed.

Soft-tissue augmentation with injectable alginate and syngeneic fibroblasts

Tissue engineering, a field that combines polymer scaffolds with isolated cell populations to create new tissue, may be applied to soft-tissue augmentation—an area in which polymers and cell populations have been injected independently. We have developed an inbred rat model in which the subcutaneous injection of a hydrogel, a form of polymer, under vacuum permits direct comparison of different materials in terms of both histologic behavior and their ability to maintain the specific shape and volume of a construct. Using this model, we compared three forms of calcium alginate, a synthetic hydrogel, over an 8-week period—standard alginate that was gelled following injection into animals (alginate post-gel), standard alginate that was gelled before injection into animals (alginate pre-gel) and alginate-RGD, to which the cell adhesion tripeptide RGD was linked covalently (RGD post-gel). Parallel groups that included cultured syngeneic fibroblasts suspended within each of these three gels were also evaluated (alginate post-gel plus cells, alginate pre-gel plus cells, and RGD post-gel plus cells). The study used 54 inbred Lewis rats (n = 9 for each of the six groups). Construct geometry was optimally maintained in the alginate post-gel group in which 58 percent of the original volume was preserved at 8 weeks and increased to 88 percent at 8 weeks when syngeneic fibroblasts were included within the gel. Volume was not as well preserved in the RGD post-gel group (25 percent of original volume at 8 weeks), but again increased when syngeneic fibroblasts were included (41 percent of original volume at 8 weeks). Maintenance of volume was poorest in the alginate pre-gel group (31 percent of original volume at 8 weeks) and failed to be augmented by the addition of fibroblasts (19 percent of original volume at 8 weeks). Histologically, the gel remained a uniform sheet surrounded by a fibrous capsule in the alginate post-gel groups. In the alginate pre-gel and RGD post-gel groups, there was significant ingrowth of a fibrovascular stroma into the gel with fragmentation of the construct. In constructs in which syngeneic fibroblasts were included, cells were visualized throughout the gel but did not extend processes or appear to contribute to new tissue formation. Material compression testing indicated that the alginate and RGD post-gel constructs became stiffer over a 12-week period, particularly in the cell-containing groups. Our results suggest that calcium alginate could be a suitable agent for soft-tissue augmentation when gelled subcutaneously following injection. The addition of syngeneic fibroblasts enhanced the ability of the gel to maintain the volume of a construct; this seems to be mediated by increased gel stiffness rather than by de novo tissue formation. Our animal model, in combination with material testing data, permits rigorous comparison of different materials used for soft-tissue augmentation.

Circular Excision of Hemangioma and Purse-string Closure: The Smallest Possible Scar

&NA; Localized cutaneous infantile hemangioma acts like a tissue expander. This rapidly growing tumor can destroy elastic fibers or cause ulceration resulting in telangiectases, cutaneous laxity, scarring, and fibrofatty residuum. Although surgeons may dispute indications and timing, most would agree that the scar of resection should be minimized. For this reason, circular excision and pursestring closure is particularly applicable for hemangioma at any stage of its evolution. The purposes of this study were to: (1) analyze the results of circular excision/purse‐string closure in all three phases of the life cycle of hemangioma; (2) quantify dimensional changes after resection; and (3) compare the scars after theoretical single‐stage lenticular excision with those after staged circular excision/pursestring closure. The authors retrospectively analyzed their experience in 25 children with localized hemangioma who underwent circular excision/purse‐string closure from 1997 to 2000. Each hemangioma was measured preoperatively and the scars were measured at most recent follow‐up (minimum, 6 months). Preoperative and postoperative dimensions were analyzed using SPSS statistical software. The study included 22 girls and three boys, with an average time to follow‐up evaluation of 13.1 months. Twenty‐one lesions were in the face and scalp, and five were in the extremity. Five tumors were resected in the proliferative phase (either because of ulceration, bleeding, or visual complications) and 21 were excised in the involuting or involuted phase. Six patients had a secondstage procedure: three had another circular excision and three had later lenticular excision. After single circular excision/purse‐string closure, the mean long‐axial diameter (length) decreased by 45 percent, the mean shortaxial width (width) decreased by 73 percent, and the mean scar area was only 15 percent of the original area. All these differences were statistically significant (p = 0.001). The average width/length ratio decreased by 50 percent, indicating a tendency for scars to linearize. There was no difference in linearization for the three phases of hemangioma (p > 0.05); extremity scars became more linear that those on the face (p = 0.01). The authors devised a formula for scar length after lenticular excision/linear closure, assuming a conventional excisional ratio of 3:1 for a circular lesion. Using this equation, the authors predicted that mean scar length after circular excision, followed by lenticular excision, would be 72 percent shorter than the calculated scar that would result from conventional lenticular excision. In three patients who underwent this two‐stage approach, the resultant scar was 69 percent shorter. Circular excision of hemangioma and purse‐string closure reduces both the longitudinal and transverse dimensions and converts a large circular lesion into a small ellipsoid scar. If subsequent revision to a linear scar is desirable, its length will be the same or slightly less than the diameter of the original lesion. No other excision and closure technique results in a smaller scar. Another advantage of this method is minimal distortion of surrounding structures. (Plast. Reconstr. Surg. 109: 1544, 2002.)

Increased Expression of Urinary Matrix Metalloproteinases Parallels the Extent and Activity of Vascular Anomalies

Objective. Matrix metalloproteinases (MMPs) and the angiogenic proteins basic fibroblast growth factor (bFGF) and vascular endothelial growth factor (VEGF) have been implicated in mechanisms of human cancer and metastasis. Assays were conducted on the urine of patients with vascular anomalies (tumors and malformations), relatively common and occasionally life-threatening disorders for which few therapies exist. We sought to determine whether these angiogenesis modulators are present in the urine and whether their expression is associated with the extent and clinical course of the vascular lesion. Methods. A total of 217 patients with vascular anomalies and 74 age-matched control subjects participated. Urinary MMP expression was determined by substrate gel electrophoresis. Urinary bFGF and VEGF levels were measured by enzyme-linked immunosorbent assay. Each patient was assigned to 1 of 2 categories (tumor or malformation) and 1 of 9 specific groups. Extent of the vascular lesion and activity were scored by a blinded clinician. Results. Urinary high molecular weight (hMW) MMPs and bFGF were significantly increased in patients with vascular tumors (53%) and vascular malformations (41%), compared with control subjects (22%). These percentages increased as a function of extent of the lesion and disease activity. hMW MMPs were increased in 4 groups: infantile hemangioma, other vascular neoplasms, lymphatic malformation and capillary-lymphaticovenous malformations, and extensive and unremitting capillary malformation and arteriovenous malformation. No significant differences among the groups were detected for low molecular weight MMPs or VEGF. Conclusions. Expression patterns of hMW MMPs and bFGF in the urine of patients with tumors and malformations are consistent with their different clinical behavior. These data represent the first evidence that MMPs are elevated in the urine of children with vascular anomalies. These data also suggest that the increased expression of urinary MMPs parallels the extent and activity of vascular anomalies in children. In addition to tumors, vascular malformations are angiogenesis dependent, suggesting that progression of a vascular malformation might be suppressed by angiogenic inhibitors, which would target bFGF and MMPs.

Reticular infantile hemangioma of the limb can be associated with ventral-caudal anomalies, refractory ulceration, and cardiac overload.

Abstract:  We describe six patients with an uncommon variant of infantile hemangioma that we have termed reticular, occurring in the extremity, which were associated with intractable ulceration, anogenito‐urinary‐sacral anomalies, and sometimes cardiac overload. The extreme end of the spectrum is exemplified by a male neonate who presented with a stained, enlarged, pulsatile lower extremity, and cardiac failure. He also had hepatic hemangiomas and ambiguous genitalia. Progressive soft tissue necrosis and bony destruction necessitated amputation. The histopathologic features differed from those of typical infantile hemangioma: infiltrative (not lobular) and involving fascia, muscle, and bone.

Tissue engineered muscle implantation for tongue reconstruction: a preliminary report.

Objectives/Hypothesis: Because current tongue reconstructive methods introduce adynamic, variably sensate tissue into the mouth, the critical functions of the tongue in articulation and deglutition may be compromised. The objective of this work was to introduce a combination of myoblasts and scaffolding material into rat hemiglossectomy defects and to examine the extent of neomuscle formation in the reconstructed area, under the hypothesis that the presence of myoblasts leads to formation of new muscle. Study Design: Randomized, prospective animal study. Methods: Myoblasts were harvested from neonatal Lewis rats, and a growth factor enriched collagen gel was prepared. Syngeneic adult animals received either hemiglossectomy alone or reconstruction with one of four experimental reconstructive preparations: collagen gel alone, collagen gel with suspended myoblasts, the gel‐cell combination in undifferentiated muscle construct form by way of tissue culture for 7 days in a preformed mold, or differentiated constructs, cultured in myoblast fusion medium. After 6 or 16 weeks, animal weight gain was recorded, animals were killed, and the tongues harvested. The tissue was examined histologically, and quality of the muscular regenerate was rated on a scale according to predefined criteria. Results: Animals in all groups gained weight appropriately. In groups receiving hemiglossectomy alone or acellular (gel only) reconstruction, there was significant scarring and lack of neomuscle formation. In groups receiving myoblast transplantation, either by way of gel suspension or in the form of undifferentiated or differentiated constructs, muscle quality was superior to controls. Conclusions: Myoblast transplantation into hemiglossectomy defects appears to lead to new muscle formation and does not inhibit normal weight gain in animals after tongue implantation.

Osteochondral reconstruction of a non-weight-bearing joint using a high-density porous polyethylene implant.

Currently, there is no reliable reconstructive modality allowing anatomic resurfacing of traumatic digital osteochondral articular defects. The purpose of the present study is to demonstrate the utility of Medpor, a high-density porous polyethylene (HDPP) scaffold biomaterial that can (1) be readily contoured to fit any joint defect, (2) permit stable internal fixation, and (3) permit osteocyte and chondrocyte ingrowth and subsequent articular cartilage resurfacing necessary to restore joint congruity. HDPP has gained wide acceptance for use in craniofacial and skeletal reconstruction and augmentation. An avian non-weight-bearing joint model was designed to study the role of the HDPP implant in small joint reconstruction. An osteochondral defect was created with a 5-mm circular punch in the humeral articular surface of both glenohumeral joints of 32 adult White Leghorn chickens. In each animal, one defect was press-fitted with a correspondingly sized HDPP implant (HDPP implant group); the contralateral defect was filled with the original osteochondral plug (isograft group) or left unrepaired (control group). At 2 weeks, and 1, 3, and 6 months,joints from each group were harvested and evaluated. Over the 6-month study period, joints in the control group demonstrated healing with dense collagenous scar tissue leaving residual defects at the articular surfaces and significant degenerative disease of the glenohumeral joints radiographically. Joints in the isograft group demonstrated near-complete resorption with some preservation of the cartilaginous cap but overall depression of the articular surface and significant degenerative joint disease. Joints in the HDPP implant group demonstrated stable fixation by highly mineralized bony trabecular ingrowth, preservation of the articular contour of the humeral head, and no evidence of significant degenerative joint disease. These findings indicate a potential role for this high-density porous polyethylene implant in the reconstruction of small joint articular and osseous defects.