Kevin J. Paik

Biomaterials for Craniofacial Bone Engineering

Conditions such as congenital anomalies, cancers, and trauma can all result in devastating deficits of bone in the craniofacial skeleton. This can lead to significant alteration in function and appearance that may have significant implications for patients. In addition, large bone defects in this area can pose serious clinical dilemmas, which prove difficult to remedy, even with current gold standard surgical treatments. The craniofacial skeleton is complex and serves important functional demands. The necessity to develop new approaches for craniofacial reconstruction arises from the fact that traditional therapeutic modalities, such as autologous bone grafting, present myriad limitations and carry with them the potential for significant complications. While the optimal bone construct for tissue regeneration remains to be elucidated, much progress has been made in the past decade. Advances in tissue engineering have led to innovative scaffold design, complemented by progress in the understanding of stem cell–based therapy and growth factor enhancement of the healing cascade. This review focuses on the role of biomaterials for craniofacial bone engineering, highlighting key advances in scaffold design and development.

Isolation of Human Adipose-Derived Stromal Cells Using Laser-Assisted Liposuction and Their Therapeutic Potential in Regenerative Medicine

Harvesting adipose‐derived stromal cells (ASCs) for tissue engineering is frequently done through liposuction. However, several different techniques exist. Although third‐generation ultrasound‐assisted liposuction has been shown to not have a negative effect on ASCs, the impact of laser‐assisted liposuction on the quality and differentiation potential of ASCs has not been studied. Therefore, ASCs were harvested from laser‐assisted lipoaspirate and suction‐assisted lipoaspirate. Next, in vitro parameters of cell yield, cell viability and proliferation, surface marker phenotype, osteogenic differentiation, and adipogenic differentiation were performed. Finally, in vivo bone formation was assessed using a critical‐sized cranial defect in athymic nude mice. Although ASCs isolated from suction‐assisted lipoaspirate and laser‐assisted lipoaspirate both successfully underwent osteogenic and adipogenic differentiation, the cell yield, viability, proliferation, and frequency of ASCs (CD34+CD31−CD45−) in the stromal vascular fraction were all significantly less with laser‐assisted liposuction in vitro (p < .05). In vivo, quantification of osseous healing by micro‐computed tomography revealed significantly more healing with ASCs isolated from suction‐assisted lipoaspirate relative to laser‐assisted lipoaspirate at the 4‐, 6‐, and 8‐week time points (p < .05). Therefore, as laser‐assisted liposuction appears to negatively impact the biology of ASCs, cell harvest using suction‐assisted liposuction is preferable for tissue‐engineering purposes.

Wound healing: an update

Wounds, both chronic and acute, continue to be a tremendous socioeconomic burden. As such, technologies drawn from many disciplines within science and engineering are constantly being incorporated into innovative wound healing therapies. While many of these therapies are experimental, they have resulted in new insights into the pathophysiology of wound healing, and in turn the development of more specialized treatments for both normal and abnormal wound healing states. Herein, we review some of the emerging technologies that are currently being developed to aid and improve wound healing after cutaneous injury.

Studies in fat grafting: Part III. Fat grafting irradiated tissue--improved skin quality and decreased fat graft retention.

Background: Following radiation therapy, skin becomes fibrotic and can present a difficult problem for reconstructive surgeons. There is an increasing belief that fat grafting under irradiated skin can reverse the damage caused by radiation. The present study evaluated the effect of fat grafting on irradiated skin, along with fat graft quality and retention rates in irradiated tissue. Methods: Nine adult Crl:NU-Foxn1nu CD-1 mice underwent 30-Gy external beam irradiation of the scalp. Four weeks after irradiation, scalp skin from irradiated and nonirradiated mice was harvested and compared histologically for dermal thickness, collagen content, and vascular density. Human fat grafts were then injected in the subcutaneous plane of the scalp. Skin assessment was performed in the irradiated group at 2 and 8 weeks after grafting, and fat graft retention was measured at baseline and every 2 weeks up to 8 weeks after grafting using micro–computed tomography. Finally, fat graft samples were explanted at 8 weeks, and quality scoring was performed. Results: Fat grafting resulted in decreased dermal thickness, decreased collagen content, and increased vascular density in irradiated skin. Computed tomographic analysis revealed significantly decreased fat graft survival in the irradiated group compared with the nonirradiated group. Histologic scoring of explanted fat grafts demonstrated no difference in quality between the irradiated and nonirradiated groups. Conclusions: Fat grafting attenuates dermal collagen deposition and vessel depletion characteristic of radiation fibrosis. Although fat graft retention rates are significantly lower in irradiated than in nonirradiated tissue, the quality of retained fat between the groups is similar.

Studies in fat grafting: Part IV. Adipose-derived stromal cell gene expression in cell-assisted lipotransfer.

Background: Fat graft volume retention remains highly unpredictable, but addition of adipose-derived stromal cells to fat grafts has been shown to improve retention. The present study aimed to investigate the mechanisms involved in adipose-derived stromal cell enhancement of fat grafting. Methods: Adipose-derived stromal cells isolated from human lipoaspirate were labeled with green fluorescent protein and luciferase. Fat grafts enhanced with adipose-derived stromal cells were injected into the scalp and bioluminescent imaging was performed to follow retention of adipose-derived stromal cells within the fat graft. Fat grafts were also explanted at days 1, 5, and 10 after grafting for adipose-derived stromal cell extraction and single-cell gene analysis. Finally, CD31 immunohistochemical staining was performed on fat grafts enriched with adipose-derived stromal cells. Results: Bioluminescent imaging demonstrated significant reduction in luciferase-positive adipose-derived stromal cells within fat grafts at 5 days after grafting. A similar reduction in viable green fluorescent protein–positive adipose-derived stromal cells retrieved from explanted grafts was also noted. Single-cell analysis revealed expression of multiple genes/markers related to cell survival and angiogenesis, including BMPR2, CD90, CD105, FGF2, CD248, TGFß1, and VEGFA. Genes involved in adipogenesis were not expressed by adipose-derived stromal cells. Finally, CD31 staining revealed significantly higher vascular density in fat grafts explanted at day 10 after grafting. Conclusions: Although adipose-derived stromal cell survival in the hypoxic graft environment decreases significantly over time, these cells provide multiple angiogenic growth factors. Therefore, improved fat graft volume retention with adipose-derived stromal cell enrichment may be attributable to improved graft vascularization.

Capillary Force Seeding of Hydrogels for Adipose-Derived Stem Cell Delivery in Wounds

Effective skin regeneration therapies require a successful interface between progenitor cells and biocompatible delivery systems. We previously demonstrated the efficiency of a biomimetic pullulan‐collagen hydrogel scaffold for improving bone marrow‐derived mesenchymal stem cell survival within ischemic skin wounds by creating a “stem cell niche” that enhances regenerative cytokine secretion. Adipose‐derived mesenchymal stem cells (ASCs) represent an even more appealing source of stem cells because of their abundance and accessibility, and in this study we explored the utility of ASCs for hydrogel‐based therapies. To optimize hydrogel cell seeding, a rapid, capillary force‐based approach was developed and compared with previously established cell seeding methods. ASC viability and functionality following capillary hydrogel seeding were then analyzed in vitro and in vivo. In these experiments, ASCs were seeded more efficiently by capillary force than by traditional methods and remained viable and functional in this niche for up to 14 days. Additionally, hydrogel seeding of ASCs resulted in the enhanced expression of multiple stemness and angiogenesis‐related genes, including Oct4, Vegf, Mcp‐1, and Sdf‐1. Moving in vivo, hydrogel delivery improved ASC survival, and application of both murine and human ASC‐seeded hydrogels to splinted murine wounds resulted in accelerated wound closure and increased vascularity when compared with control wounds treated with unseeded hydrogels. In conclusion, capillary seeding of ASCs within a pullulan‐collagen hydrogel bioscaffold provides a convenient and simple way to deliver therapeutic cells to wound environments. Moreover, ASC‐seeded constructs display a significant potential to accelerate wound healing that can be easily translated to a clinical setting.

Studies in Fat Grafting: Part V. Cell-Assisted Lipotransfer to Enhance Fat Graft Retention is Dose Dependent

Background: Cell-assisted lipotransfer has shown much promise as a technique for improving fat graft take. However, the concentration of stromal vascular fraction cells required to optimally enhance fat graft retention remains unknown. Methods: Human lipoaspirate was processed for both fat transfer and harvest of stromal vascular fraction cells. Cells were then mixed back with fat at varying concentrations ranging from 10,000 to 10 million cells per 200 &mgr;l of fat. Fat graft volume retention was assessed by means of computed tomographic scanning over 8 weeks, and then fat grafts were explanted and compared histologically for overall architecture and vascularity. Results: Maximum fat graft retention was seen at a concentration of 10,000 cells per 200 &mgr;l of fat. The addition of higher number of cells negatively impacted fat graft retention, with supplementation of 10 million cells producing the lowest final volumes, lower than fat alone. Interestingly, fat grafts supplemented with 10,000 cells showed significantly increased vascularity and decreased inflammation, whereas fat grafts supplemented with 10 million cells showed significant lipodegeneration compared with fat alone Conclusions: The authors’ study demonstrates dose dependence in the number of stromal vascular fraction cells that can be added to a fat graft to enhance retention. Although cell-assisted lipotransfer may help promote graft survival, this effect may need to be balanced with the increased metabolic load of added cells that may compete with adipocytes for nutrients during the postgraft period.

Studies in fat grafting: Part I. Effects of injection technique on in vitro fat viability and in vivo volume retention.

Background: Fat grafting has become increasingly popular for the correction of soft-tissue deficits at many sites throughout the body. Long-term outcomes, however, depend on delivery of fat in the least traumatic fashion to optimize viability of the transplanted tissue. In this study, the authors compare the biological properties of fat following injection using two methods. Methods: Lipoaspiration samples were obtained from five female donors, and cellular viability, proliferation, and lipolysis were evaluated following injection using either a modified Coleman technique or an automated, low-shear device. Comparisons were made to minimally processed, uninjected fat. Volume retention was also measured over 12 weeks after injection of fat under the scalp of immunodeficient mice using either the modified Coleman technique or the Adipose Tissue Injector. Finally, fat grafts were analyzed histologically. Results: Fat viability and cellular proliferation were both significantly greater with the Adipose Tissue Injector relative to injection with the modified Coleman technique. In contrast, significantly less lipolysis was noted using the automated device. In vivo fat volume retention was significantly greater than with the modified Coleman technique at the 4-, 6-, 8-, and 12-week time points. This corresponded to significantly greater histologic scores for healthy fat and lower scores for injury following injection with the device. Conclusion: Biological properties of injected tissues reflect how disruptive and harmful techniques for placement of fat may be, and the authors’ in vitro and in vivo data both support the use of the automated, low-shear devices compared with the modified Coleman technique.

Cell‐Assisted Lipotransfer Improves Volume Retention in Irradiated Recipient Sites and Rescues Radiation‐Induced Skin Changes

Radiation therapy is not only a mainstay in the treatment of many malignancies but also results in collateral obliteration of microvasculature and dermal/subcutaneous fibrosis. Soft tissue reconstruction of hypovascular, irradiated recipient sites through fat grafting remains challenging; however, a coincident improvement in surrounding skin quality has been noted. Cell‐assisted lipotransfer (CAL), the enrichment of fat with additional adipose‐derived stem cells (ASCs) from the stromal vascular fraction, has been shown to improve fat volume retention, and enhanced outcomes may also be achieved with CAL at irradiated sites. Supplementing fat grafts with additional ASCs may also augment the regenerative effect on radiation‐damaged skin. In this study, we demonstrate the ability for CAL to enhance fat graft volume retention when placed beneath the irradiated scalps of immunocompromised mice. Histologic metrics of fat graft survival were also appreciated, with improved structural qualities and vascularity. Finally, rehabilitation of radiation‐induced soft tissue changes were also noted, as enhanced amelioration of dermal thickness, collagen content, skin vascularity, and biomechanical measures were all observed with CAL compared to unsupplemented fat grafts. Supplementation of fat grafts with ASCs therefore shows promise for reconstruction of complex soft tissue defects following adjuvant radiotherapy. Stem Cells 2016;34:668–673

Studies in fat grafting: Part II. Effects of injection mechanics on material properties of fat.

Background: Although fat grafting can address many soft-tissue deficits, results remain inconsistent. In this study, the authors compared physical properties of fat following injection using an automated, low-shear device or the modified Coleman technique. Methods: Lipoaspirate was obtained from nine patients and processed for injection using either a modified Coleman technique or an automated, low-shear device. Fat was passed through a 2-mm cannula and compared with minimally processed fat. A rheometer was used to measure the storage modulus and shear rate at which tissues began to lose their solid-like properties. Viscosity was also measured, and gross properties of treatment groups were evaluated qualitatively with a glass slide test. Results: Fat injected through an automated, low-shear device closely matched physical properties of minimally processed fat. The storage modulus (G′) of fat for the device group was greater than for the modified Coleman group, and the onset of breakdown was delayed. Similarly, viscosity measurement of fat from the automated device closely matched minimally processed fat and was greater than that of othe modified Coleman group. Conclusions: The physical properties of lipoaspirate processed using an automated, low-shear device with a 2-mm cannula preserved the intactness of fat more than the modified Coleman technique. The authors’ rheologic data demonstrate less damage using an automated device compared with the modified Coleman technique and potentially support its use for improved fat graft integrity.