Jay W. Calvert

Characterization of osteoblast-like behavior of cultured bone marrow stromal cells on various polymer surfaces

The creation of novel bone substitutes requires a detailed understanding of the interaction between cells and materials. This study was designed to test certain polymers, specifically poly(caprolactone) (PCL), poly(D,L-lactic-CO-glycolic acid) (PLGA), and combinations of these polymers for their ability to support bone marrow stromal cell proliferation and differentiation. Bone marrow stromal cells were cultured from New Zealand White rabbits and were seeded onto glass slides coated with a thin layer of PCL, PLGA, and combinations of these two polymers in both a 40:60 and a 10:90 ratio. Growth curves were compared. At the end of 2 weeks, the cells were stained for both matrix mineralization and alkaline phosphatase activity. There was no statistically significant difference in growth rate of the cells on any polymer or polymer combination. However, there was a striking difference in Von Kossa staining and alkaline phosphatase staining. Cells on PCL did not show Von Kossa staining or alkaline phosphatase staining. However, in the 40:60 and 10:90 blends, there was both positive Von Kossa and alkaline phosphatase staining. These data indicate that PCL alone may not be a satisfactory material for the creation of a bone substitute. However, it may be used in combination with PLGA for the creation of a bone substitute material.

Survival of diced cartilage grafts: an experimental study.

Background: Use of diced cartilage grafts in rhinoplasty surgery has recently undergone a dramatic resurgence. Some authors recommend wrapping diced cartilage with oxidized methylcellulose (i.e., Surgicel). Others prefer wrapping diced cartilage with autogenous deep temporal fascia. This study was designed to compare the behavior of diced cartilage grafts as an isolated entity or when wrapped with either Surgicel or deep temporal fascia. Methods: Septal cartilage and deep temporal fascia were obtained from five patients in this institutional review board–approved study. The cartilage was diced into 0.5-mm pieces and implanted into subcutaneous dorsal skin pockets of Rowlett nude rats as isolated diced cartilage grafts, or wrapped in Surgicel or deep temporal fascia. Pieces of Surgicel and fascia were implanted alone as controls. The specimens were harvested at 8 weeks; processed by thin-section histology; stained with hematoxylin and eosin, Masson’s trichrome, glial fibrillary acidic protein, safranin-O, and Evans van Gieson; and evaluated to determine cartilage viability and architectural characteristics. Results: Diced cartilage wrapped in Surgicel yielded the lowest percentage viability and minimal staining with hematoxylin and eosin, trichrome tissue, safranin-O, and Evans van Gieson stains. Diced cartilage grafts wrapped in fascia had the greatest percentage of viable cartilage. The grafts wrapped in deep temporal fascia also demonstrated the strongest staining with the aforementioned stains. Differences in uptake of glial fibrillary acidic protein were not noticeable between the three groups. However, absolute numbers of nucleated lacunae and basophilic lacunae were significantly higher for grafts wrapped in deep temporal fascia. Conclusions: Diced cartilage grafts have reemerged as a viable method for nasal reconstruction in both primary and secondary rhinoplasty. Wrapping diced cartilage specimens contains the individual pieces and facilitates graft placement. Surgicel wraps appear to incite an inflammatory response and subsequent absorption of the cartilage. Fascia wraps appear to minimize inflammatory responses to the cartilage, thereby preserving healthy cartilage. This study demonstrates that deep temporal fascia is the preferred envelope with which to facilitate graft containment and maintain chondrocyte viability of diced cartilage grafts.

New frontiers in bone tissue engineering

No single scientific field can generate the ideal method of engineering bone. However, through collaboration and expansion of programs in bone tissue engineering, the right combination of materials, cells, growth factors, and methodology will come together for each clinical situation such that harvesting bone grafts will become obsolete. This article reviews the need for engineered bone and provides a historical perspective of bone engineering research, current research efforts, and the future direction of this work.

The evolution of the midface lift in aesthetic plastic surgery.

Summary: The midface lift has recently gained significant popularity with many surgeons. It allows the surgeon an opportunity to achieve greater facial harmony with facial rejuvenation procedures by correcting midfacial atrophy, addressing the tear trough deformity, and correcting the perceived malposition of the malar fat pad. This article examines the history of midfacial procedures. Surgical attempts at improving the aging face have evolved from minimal excisions and skin closure to aggressive dissections at multiple planes. The midface target area is peripheral to classic approaches, and its correction has required further anterior dissection from a distance or direct access centrally. Ultimately, conquering the stigmata of midface aging is entirely related to vectors and volume.

Mandibular reconstruction: Are two flaps better than one?

This study compared the combined iliac and ulnar forearm flaps with the osteomusculocutaneous fibular free flap for mandibular reconstruction. A retrospective study of 40 patients who had oromandibular reconstruction was performed, of whom 23 patients had a combined iliac crest without skin and ulnar forearm free flap. Seventeen patients had an osteomusculocutaneous free fibular flap. Ten women and 30 men with a mean age of 57.5 years comprised this study population. Ninety percent of the cases were squamous cell carcinoma (55%, T4), of which 11% were recurrent tumors. Anterolateral mandibular defects constituted 52.9% of the fibular reconstructions and 60.9% accounted for the iliac/ulnar reconstructions. The mean bone gaps were 8.79 cm and 8.95 cm respectively. Functional evaluation was based on the University of Washington Questionnaire through phone calls and personal communication. The mean hospital stay was 15.43 days and 10.09 days for the fibular and iliac/ulnar flaps respectively. The facial artery (64.7%) and facial vein (60%) were the main recipient vessels for the fibular reconstructions whereas the external carotid artery (95.6%) and the internal jugular vein (66.7%) were the main recipient vessels for the iliac/ulnar reconstruction. Overall flap survival was 96.8% (100% of fibular flaps and 95.65% of iliac/ulnar flaps). Two flaps were lost in the iliac/ulnar series because of unsalvageable venous thrombosis. Local complications for the iliac/ulnar flaps were 30.4% but were 5.9% for the fibular reconstructions. Function such as speech, swallowing, and chewing were notably better in the fibular than the iliac/ulnar group in 23 of the patients tested. The cosmetic acceptance of 77.8% of the fibular flaps was judged to be excellent and good, whereas 71.4% of the iliac/ulnar flaps were rated good. It appears that within this study population the free osteomusculocutaneous fibular flap had fewer local complications and a higher flap survival rate than the combined iliac/ulnar forearm flaps. Overall functional outcome was also improved. The use of the double flap may be appropriate in massive oromandibular defects, but may be less appropriate in more modest functional reconstructions of mandibular defects.

Osteoblastic phenotype expression of MC3T3-E1 cells cultured on polymer surfaces.

Background: Current efforts in bone tissue engineering have as one focus the search for a scaffold material that will support osteoblast proliferation, matrix mineralization, and, ultimately, bone formation. The goal is to develop a bone substitute that is functionally equivalent to autograft bone. Previously published reports have shown that osteoblasts exhibit varying rates and degrees of proliferation and mineralization when grown on different surfaces. Methods: This study presents a histologic and biomolecular analysis of MC3T3-E1 murine preosteoblast cells grown on poly(lactide-co-glycolide) (PLGA) versus poly(-caprolactone) (PCL), two commonly studied scaffold polymers. MC3T3-E1 cells were cultured on slides coated with either PLGA or PCL, and on uncoated glass slides as control, with six slides in each group. After 6 weeks in culture, the cells were stained for osteocalcin, alkaline phosphatase activity, and matrix mineralization. In addition, to assess the effects of the surface material on phenotypic expression at the molecular level, MC3T3-E1 cells were cultured on polymer-coated 24-well plates for 4 days, and analyzed by reverse transcription polymerase chain reaction for the expression of osteocalcin and alkaline phosphatase. Results: The results showed that three groups of slides stained positively for osteocalcin at 6 weeks. However, markedly less alkaline phosphatase activity and mineralization were observed on the cells grown on PCL. Real-time polymerase chain reaction assays subsequently revealed decreased expression of both markers by cells cultured on PCL compared with PLGA. Conclusions: These results suggest that PCL does not support the full expression of an osteoblastic phenotype by MC3T3-E1 cells. PCL, therefore, is less desirable as a scaffold polymer in bone tissue engineering in so far as supporting bone formation is concerned. However, because PCL has favorable handling characteristics and strength, modifications of PCL may prompt further investigation.

Release Kinetics of Polymer-bound Bone Morphogenetic Protein-2 and Its Effects on the Osteogenic Expression of Mc3t3-e1 Osteoprecursor Cells

Background: In an effort to augment scaffold performance, additives such as growth factors are under investigation for their ability to optimize the “osteopotential” of synthetic polymer scaffolds. In parallel research, bone morphogenetic protein-2 (BMP-2), a growth factor that initiates bone formation, has been locally delivered to augment fracture healing and spinal fusion. The authors hypothesize that BMP-2 can be covalently bound to a polymer substrate, increasing its concentration and bioavailability over longer periods, thus improving the efficacy of the growth factor and subsequently the bony matrix production. It would remain bound longer when compared with published controls. This prolonged binding would then increase the bioavailability of the growth factor and thus increase bony matrix production over a longer interval. Methods: Mouse preosteoblast MC3T3-E1 cells were cultured on poly(lactic-co-glycolic acid) and polycaprolactone polymer disks covalently bound with BMP-2 to assess the progression and quality of osteogenesis. Covalent binding of BMP-2 to each polymer was visualized by immunohistochemical analysis of polymer-coated microscope slides. The quantity of covalently bound BMP-2 was determined using enzyme-linked immunosorbent assay. Results: Polymerase chain reaction results showed elevated expression levels for alkaline phosphatase and osteocalcin genes. BMP-2 was released from polycaprolactone over 2 weeks, with 86 percent remaining covalently bound, in contrast to 93 percent retained by poly(lactic-co-glycolic acid). Conclusions: BMP-2, proven to alter polymer osteogenicity, remained bound to poly(lactic-co-glycolic acid), which may render poly(lactic-co-glycolic acid) an ideal choice as a polymer for scaffold-based bone tissue engineering using growth factor delivery.

Autogenous dorsal reconstruction: maximizing the utility of diced cartilage and fascia.

The problem of reconstructing the dorsum of the nose is complex and a source of frustration for both patients and surgeons. Dorsal deficiencies due to various etiologies and the need for dorsal contouring cause the plastic surgeon to look to time-honored techniques such as osseocartilaginous rib grafts while also searching for other options that may be less technically challenging and have the benefit of temporal success. Diced cartilage wrapped with deep temporal fascia is just such a method to achieve reliable dorsal reconstructions. The various ways to use diced cartilage and deep temporal fascia are discussed.

Pharmacologic enhancement of rat skin flap survival with topical oleic acid.

This study was instituted to investigate in a rat model the effect of topical coadministration of the penetration enhancer oleic acid (10% by volume) and RIMSO-50 (medical grade dimethyl sulfoxide, 50% by volume) on rat skin flap survival. A rectangular abdominal skin flap (2.5 × 3 cm) was surgically elevated over the left abdomen in 40 nude rats. The vein of the flaps neurovascular pedicle was occluded by placement of a microvascular clip, and the flap was resutured with 4-0 Prolene to its adjacent skin. At the end of 8 hours, the distal edge of the flap was reincised to gain access to the clips and the clips were removed. After resuturing of the flaps distal edge to its adjacent skin, the 40 flaps were randomly divided into four groups. Group 1 (control) flaps were treated with 5 g of saline, group 2 (dimethyl sulfoxide) flaps were treated with 2.7 g of dimethyl sulfoxide (50% by volume), group 3 flaps (oleic acid) were topically treated with 0.45 g of oleic acid (10% by volume), and group 4 (dimethyl sulfoxide plus oleic acid) flaps were treated with a mixture of 0.45 g of oleic acid (10% by volume) and 2.7 g of dimethyl sulfoxide (50% by volume) diluted in saline. Each flap was topically treated with 5 ml of drug-soaked gauze for 1 hour immediately after clip removal to attenuate reperfusion injury. Thereafter, drug was applied topically once daily for 4 more days. Digital photographs of each flap were then taken on day 6 and the flaps were then harvested. The percentage of skin survival in each flap was determined by computerized morphometry and planimetry. The mean surviving area of group 3 (oleic acid–treated flaps) was 23.60 ± 4.19 percent and was statistically higher than that in group 1 (control, saline-treated flaps) at 7.20 ± 2.56 percent. The mean surviving area of group 2 (dimethyl sulfoxide–treated flaps) at 18.00 ± 5.23 percent and group 4 (oleic acid–and dimethyl sulfoxide-treated flaps) at 9.90 ± 3.44 percent did not achieve statistically higher mean surviving areas than controls. A topical solution of oleic acid (10% by volume) caused a statistically significant increase in the survival of rat abdominal skin flaps relative to controls. Dimethyl sulfoxide and the two experimental drugs together did not increase the percentage of flap survival when given as a single 5-ml dose released from a surgical sponge at reperfusion for 1 hour and then daily for a total of 5 days. The reasons for the lack of response are unknown but may have included the technical difficulty of delivering an adequate dose of dimethyl sulfoxide topically and immiscibility between dimethyl sulfoxide and oleic acid. Further studies may be warranted.

Reconstructive rhinoplasty: operative revision of patients with previous autologous costal cartilage grafts.

Background: Costal cartilage grafts are used more frequently in secondary rhinoplasty. However, these procedures result in patients that require further revision operations that are not trivial. The purpose of this study was to assess the outcomes of reconstructive rhinoplasty performed on patients with a history of prior secondary or tertiary rhinoplasty using autogenous costal cartilage grafts. Methods: A retrospective review was conducted of tertiary rhinoplasty procedures. Outcomes of interest included the indications for the revision operation; revision rate following the tertiary costal cartilage revision; whether or not the use of costal cartilage grafts for the reconstructive rhinoplasty was a planned event; and perioperative incidents such as infection, structural collapse, and scarring requiring further surgery. Results: Forty-six patients met inclusionary criteria. The revision rate following tertiary rhinoplasty performed by the primary author was found to be 24 percent (11 of 46). The reasons for these revision operations were nasal airway obstruction (n = 6), aesthetic dissatisfaction (n = 4), and infection (n = 1). Outcomes of the revisions were satisfactory, with no adverse complications requiring further surgery. Conclusions: Reconstructive rhinoplasty may be required in patients who have undergone previous costal cartilage grafting for secondary rhinoplasty. These revision operations can be performed successfully to help patients achieve both improved function and aesthetic results but have a higher than usual revision rate themselves. The revisions of these operations can be managed with standard revision operations that result in satisfactory outcomes. CLINICAL QUESTION/LEVEL OF EVIDENCE: Therapeutic, IV.