TISSUE ENGINEERING IN PLASTIC SURGERY – WHAT HAS BEEN DONE

INTRODUCTION

Tissue engineering (TE) was firstly defined as “the application of the principles and methods of engineering and the life sciences towards the fundamental understanding of structure–function relationships in normal and pathologic mammalian tissues and the development of biologic substitutes that restore, maintain, or improve tissue function” ^1,2. This relies on interdisciplinary collaboration between cell biologists, material engineers, biotechnologists, various medical specialties and industry manufacturers³.

TE and Plastic surgery share the common goal of restoring structure and function⁴. TE has the potential to reduce morbidity of the current approaches by eradicating donor site lesions, reducing hospital stay and the associated risks and costs³. Classic TE approaches encompass three components for regeneration: cells to form a matrix, a scaffold for transplantation and temporary support, and environmental factors^2,3,5–8.

Possible cell sources include embryonic stem cells, adult stem cells and differentiated cells^3,5,6,8,9. Embryonic stem cells are totipotent; however, they are difficult to obtain and raise ethical objections and concerns of tumorigenicity³. Mesenchymal stem cells (MSC) can be pluripotent or multipotent, they can be tissue derived, are available in large quantities and are increasingly recognized as the preferable cell source^3,8. Induced pluripotent stem cells discovered by Takahashi and Yamanaka¹⁰ made possible the use of somatic cells in tissue engineering by reversing their differentiation state into pluripotent cells, but their use is still at its infancy^3,8,9.

Scaffolds are designed to guide and support cells, allowing a physiologic three-dimensional proliferation and differentiation^5–9. They can be biological or synthetic, depending on the fabrication materials. Furthermore, they can be solid, which requires surgical implantation, or can exhibit gel form, which – when injected – acquire the appropriated form of the defect in situ^3,6.

Environmental factors consist of bioactive molecules (growth factors and cytokines), oxygen tension and mechanical or electrical stimulation, that alter cell’s response, improving wound healing and regeneration^3,5,7,9,11.

This review will present an overview of published literature regarding tissue engineering in plastic surgery in clinical and pre-clinical studies, its current cells’ sources and applications, its disadvantages and future perspectives.

The aim of this work is to create awareness regarding one of the medicine’s greatest evolutions and the importance of implementation into practice of these new methods in reconstructive and aesthetic plastic surgery.

USES OF TISSUE ENGINEERING IN PLASTIC AND RECONSTRUCTIVE SURGERY

Tissue engineering is the promising alternative to the current plastic and reconstructive surgery approaches and has yielded small successes so far.

Below, a brief outline of the various current and potential therapeutic applications of tissue engineering, the multiple tissue sources and their uses (Figure 1), are presented.

SKIN DEFECTS

The traditional autologous and allogeneic epidermal sheets have been proven successful in wound repair. However, they are limited in treating extensive wounds and fail to prevent wound contraction¹².

There are multiple bioengineered products used in skin defects (Table 1). These derivatives, besides their indications, vary in administration, preservation, shelf life (acellular derivatives have longer shelf life, lasting years) and host immune response (present with porcine or bovine derivatives)^9,13,14.

Topical therapies have the ability to promote wound vascularization promotion, reduce wound contraction and induce keratinization^9,15. For example, recombinant human granulocyte/macrophage colony-stimulating factor (GM-CSF) topically applied had positive effects on venous and diabetic ulcers of the lower limb¹⁶. Also, Regranex, recombinant human PDGF, decreases wound closure time and increases complete wound healing⁹; this topical gel is used on diabetic neuropathic ulcers and advanced stage pressure ulcers, with great results, but as a growth factor, its application caries risk of malignancy^9,16.

Adipose tissue is a popular source of stem cells for wound healing^15–19. The paracrine effect of adipose-derived stem cells (ADSC) stimulates the secretion of cytokines with anti-inflammatory properties and induces skin neovascularization. Furthermore, they recruit endogenous stem cells and have secretory effects over dermal fibroblasts and keratinocytes^15,19. ADSC suspension in saline and platelet rich plasma (PRP) injected showed to be particularly successful in cases with small areas of skin necrosis after hyaluronic acid filler injection^18,20. In Korea, this is widely used over hyaluronidase, and is very effective in the relief of local ischemia and pain^18,20.

Autologous ADSC administered by repeated hypoinvasive computer-assisted injections showed systematic improvement or remission in all 20 patients treated for chronic ulcers caused by radiotherapy¹⁷.

Immersion of scaffolds in an ADSC cell suspension has been used for the treatment of recurrent or chronic cutane­ous lesions, such as diabetic or chronic radiation ulcers¹⁸. As an example, Terudermis is used as an artificial dermis that must be sutured to adjacent skin as a protective sur­face. This approach may be an option for small traumatic defects or skin cancers to avoid the use of local flaps¹⁸.

Cell sheet engineering, is a promising alternative that consists in a scaffold-free tissue-engineered product obtained through the culture of ADSC or human keratinocytes, dermal fibroblasts and dermal microvascular endothelial cells on a thermoresponsive surface or a standard cell culture surface^12,15. Studies conducted on mice showed a short culture time period, great stability, high cell survival, high cell residence time and a stable neovascularization, which impacts on full-thickness skin regeneration^12,15.

The use of ADSC combined with Platelet-rich plasma (PRP) potentiate adipogenesis and graft maintenance, especially in association with insulin¹⁹. PRP is a small volume of plasma concentrated with autologous human platelets and growth factors, it is currently used in regenerative medicine due to its capacity to stimulate tissue regeneration^16,17,21.

PRP is indicated in a mixture with fat for chronic or post-traumatic ulcers and loss of substance of the lower limbs^16,17,21. PRP used independently in a gel form or as an injection demonstrated great results in chronic/non-healing cutaneous ulcers, acute limb soft tissue wounds, trauma wounds and to stop bleeding in surgical flaps^16,21,22. Possible associated risks include recurrence and growth of a pre-existing tumour¹⁶.

As mentioned above, keratinocytes and fibroblasts are used in several tissue-engineered constructs. These constructs have several indications as aforementioned, but it is still unclear when they should be indicated over a skin graft⁹.

Deformities associated with burn injury are frequently treated by plastic surgery procedures. Their incidence is increasing and they can lead to deadly consequences like burn shock and sepsis^23,24. Currently, burn wounds’ treatment is based on the removal of devitalized tissue and coverage with an autologous split-thickness skin graft, but if the burned surface area is large this technique loses its value²³. Temporary skin substitutes consist of biologically active patches that provide protection from infection and trauma, and provide pain control, while re-epithelialization occurs²³. For example, Integra Dermal Regeneration Template is commonly used as a temporary approach in burn reconstruction, consisting of a layer of collagen and glycosaminoglycan covered by a semipermeable silicone⁹. Nowadays, as mentioned, there are already several bioengineered permanent skin substitutes in burn healing, including the FDA approved substitute, for treatment of severely burned patients – Epicel ^9,13. Apligraf and Dermagraft need to undergo more clinical trials to evaluate their potential in burn injuries^7,13.

Scarring is incomplete healing that leaves some deformation and/or defect¹⁸. Scars can affect negatively the appearance, can engender psychological illness, low self-esteem and isolation. ADSC therapy can be used to modulate scar formation. These cells injected repeatedly into the scarred tissue bed cause the scar to become soft, altering its remodelling process, which may avoid secondary surgery¹⁸. Although there is no scientific data published, ADSC-based cell therapy is used for prevention of scaring in severe facial trauma¹⁸. In clinical settings, if the healing process of an extensive wound will predictively take more than 2 weeks, skin grafting is advisable to minimize scarring²⁵. There are two skin substitutes constructs indicated for scar reconstruction: SureDerm (acellular human cadaveric dermis) and Integra, used in burn scar contractures and keloid scars reconstructions²⁵.

Researchers believe that, by solving how to rapidly revascularize grafts/substitutes, the acceleration of the wound healing process will limit scar formation²⁵. The goal for TE is therefore the generation of prevascularized constructs able to be transplantable with standard microsurgical anastomotic techniques. Klar et al. ²⁶, for the first time, used adipose tissue’s stromal vascular fraction (SVF) for assembling a capillary plexus, for anastomosis with the recipient’s circulation, and transplanted it onto immune-deficient rats. This was beneficial in achieving an increased graft dimension and absence of shrinkage²⁶.

In the lab: At the 3B’s Research Group, alternative methodologies for wound healing are being explored. Innovative scaffolds such as spongy-like hydrogels were shown to improve healing outcomes in animal^27–30, also by promoting neovascularization. The same was verified using cell sheet engineering, a scaffold-free strategy that uses sheets of cells, being a completely biologic approach (Figure 2)^12,15.

ADIPOSE TISSUE

Adipose derived stem cells have similar differentiation potential as bone marrow-derived MSC. They are easily harvested from lipoaspirates through enzymatic dissociation (with e.g. collagenase) and isolation from the SVF^4,7,8,13,31. ADSC can then be used as a cell suspension, mixed with aspirated fat or seeded onto scaffolds⁸. Minonzio et al.³¹ demonstrates that these cells can be preserved frozen without losing their ability to differentiate. This justifies the existence of a new business of ADSC banking¹⁸.

Fat grafting is a frequent procedure used for soft tissue filling, but it has the disadvantage of significant absorption (40% to 80%) of the transplanted fat^8,13,32. Contour deformities can cause both aesthetic and functional problems³². Many permanent (e.g. silicone, polymethyl-methacrylate) and temporary (e.g. HA, collagen) fillers are used, however, they often have poor results and complications³³. ADSC in combination with fat grafting are indicated for volume restoration, because of their capacity to increase fat tissue survival rates, promote higher connective tissue formation, decrease necrotic tissue and improve patient satisfaction^32,34. Bashir et al.³² was the first to report clinically the use of ex vivo expanded ADSC-enriched fat graft for facial lipodystrophy^13,35,36. This combination use was also reported in lower limb atrophy from critical limb ischemia^13,35 and chronic ulcers caused by radiation therapy^16,17.

Fat grafts enriched with ADSC were also used for breast augmentation, with cosmetic or cancer reconstruction purposes^{8,13,17,36–38}. Their application resulted in breast’s circumference enlargement, with minimal absorption and reduced complications^39,40. However, these cells secrete proangiogenic growth factors, that may result in an increased breast cancer’s metastatic risk³⁷. ADSC were found to increase growth of active tumour cells but not resting tumour cells⁴¹, while other study demonstrated that the risk depended on the delivery methods⁴². In contrast, there are also reports of a decrease in tumour growth and metastasis index^37,43.

Inoculation of Crohn’s disease fistulas external openings with autologous ADSC was successful in 6 of 20 patients in a pilot study^16,17. Since then, this technique was used to repair tracheomediastinal fistulas caused by cancer ablation^16,17,44.

ADSC, due to their immunomodulatory and anti-inflammatory effects, were used in other autoimmune diseases such as systemic lupus erythematosus, autoimmune arthritis, rheumatoid arthritis and acute graft-versus-host disease¹⁶. They also have applications for neural regeneration in Parkinson’s and Alzheimer’s disease^16,19; hepatic regeneration¹⁶; and mixed with PRP for age-related macular degeneration and corneal epithelium repair^13,16,17,19.

CRANIOCERVICAL DEFECTS

Head and neck structures control several senses, vital functions, the swallowing process, communication, facial animation and aesthetics⁴⁵. Craniofacial defects can be congenital (e.g. cleft lip, calvarial defects or Romberg syndrome) or acquired (e.g. trauma or oncological resection)^45–47. The functional, aesthetic and social effects resulting from these anomalies can severally affect the patient’s quality-of-life^45,46.

Soft tissue defects causing facial contour deformities require soft tissue augmentation therapies that usually consist of autologous fat grafting³². Case reports and prospective studies showed that ADSC enriched fat is more beneficial for facial contour deformities repair, reducing the need to repeat the grafting^32,34. This combination is indicated for loss of substance on the face, volume loss in aging, hemifacial microsomia, scleroderma, Parry-Romberg syndrome and in facial skin necrosis^16–18. PRP therapies have shown benefits in deep-plane rhytidectomy (for reducing tissue inflammation, oedema and ecchymosis)^48–50, Romberg syndrome and facial tissue atrophy type 1 and 2 ^51–54.

Cranial bone defects are extremely common in children⁵⁵. In craniofacial bone’s replacement higher porosity is essential for vascularization, osteoblast proliferation and migration, but this leads to lower mechanical properties⁴⁶. For bone replacement, it is thought to be essential to incorporate mineral phases onto the scaffolds for osteoinductivity^46,56. Calvarial defects are usually treated with cranioplasty using autologous bone grafting, titanium mesh implants or polymethylmethacrylate⁴⁵. Several animal and human studies showed promising results with the use of ADSC in tissue engineered constructs for these defects^{9,13,16,17,45,57}. Case reports demonstrate new bone formation using ADSC in combination with an autologous cancellous bone from the iliac crest and fibrin glue for calvarial traumatic defects, without using exogenous growth factors^57,58. The benefits of ADSC use were also reported in mandibular, maxilla, frontal sinus and nasal septum defects⁵⁹.

Bone marrow-derived MSC were beneficial on the improvement of bone defects healing⁶⁰ and have been used for a functional neomandible creation⁶¹.

There is also a FDA approved product for alveolar ridge augmentation, including in alveolar clefts, INFUSE bone graft, that consists of a collagen sponge soaked with recombinant human BMP-2⁶².

Cartilage tissue engineering could provide adequate amounts of tissue to overcome the cartilage donor site shortage and morbidity^45,47. Nasal lesions often occur after oncologic resection of skin cancers, this resection is usually very mutilating requiring multiple local skin flaps⁴⁷. Injected gelatinous chondroid matrix with chondrocytes harvested from the auricular cartilage, was used for nasal augmentation, with formation of hard neocartilage and satisfactory long-lasting effects⁶³. In other human trial, resected alar cartilage was replaced with a tissue engineered mesh; however, the authors were incapable of proving the cartilaginous and not scar nature of the neotissue⁶⁴.

One of the biggest challenges in plastic surgery is auricle reconstruction, due to acquired or congenital deformities (e.g. microtia and prominent ears) ^4,65. The current treatments for these defects include an auricular shaped autologous rib cartilage or artificial implants^4,45,47,65. One human trial, including 4 patients with microtia, showed, with great results, the possibility of injecting cultured chondrocytes into the subcutaneous pocket on the fascia of the lower abdomen to form a neocartilage block that was surgically harvested and carved into the shape of the auricle and implanted⁶⁶. TE in nasal and auricle reconstruction is far advanced in animal studies, but still lacks human trials; there are high hopes for cartilage culture methods of chondrocytes or pluripotent stem cells on 3D scaffolds^4,45,47,65.

Tracheal defects can be life-threatening. The current synthetic materials that have been used as tracheal substitutes present a risk of granulation formation or stenosis, and allografts imply the need for lifelong immunosuppression⁴⁷. There are several tissue- engineered substitutes, such as decellularized tracheal allografts^9,47,67,68, a rigid prosthetic tube lined by a free radial forearm flap^9,68,69, a decellularized tracheal autograft with bone marrow MSC and bronchial stem cells^11,70, and a tracheal allograft combined with a buccal mucosal graft^9,68,71. A case report of the use of a decellularized tracheal autograft repopulated with recipient’s respiratory epithelium and mesenchymal stromal cells on a 10-year-old child showed that the graft was completely integrated, but there was no proof of neocartilage formation and it didn’t grow like the adjacent native trachea^47,70.

The laryngeal structures require a complex neural control and integration into reflex movement patterns. Therefore, tissue engineered laryngeal substitutes are still in a preclinical stage, since vocal fold motion has not yet been achieved⁴⁷.

MUSCULOSKELETAL TISSUES

The actual gold standards for bone defects are autologous bone grafting and cadaveric/decellularized bone allografts^5,9. These methods are limited by variable graft resorption^72,73, risk of graft infection⁷² and the potential of disease transmission^9,73.

Bone TE has been applied for years, in simpler forms such as corticotomy for osteoinduction⁹. Nowadays, the tissue engineered methods include transplantation of in vitro cultivated cells and guided tissue regeneration with bone mass or artificial material⁴. Osteoblasts, MSC, embryonic and skeletal muscle stem cells, all present osteogenic potential⁸. Scaffolds with calcium phosphate, hydroxyapatite, silica and collagen as the matrix are seen as necessary⁸.

The first reported tissue engineered bone repair was in 2001. Bone marrow MSC were used in 3 patients to treat large bone defects, from the tibia, ulna and humerus⁷⁴. The INFUSE bone graft, used for alveolar clefts repair, can also be used for open tibial shaft fractures⁶². A similar product, OP-1 Implant, consisting of recombinant human BMP-2 with a bovine collagen carrier, was FDA approved for long bones non-union refractory lesions^9,75. MSC transplantations improved hematopoietic stem cell engraftment for healing critical sized bone defects and for children with osteogenesis imperfecta^11,61.

The current treatments for articular cartilage injury are essentially for symptoms’ control¹⁹. Bone marrow MSC, cultured in a collagen gel scaffold, are useful in TE for intervertebral disk replacement, knee joint resurfacing and digital-joint engineering^5,11,76,77. These cells were also used to repair articular defects with intra-articular injections⁷⁸. These treatments showed efficiency in reducing pain and improving walking ability¹³. Other treatment alternative is expanded autologous chondrocytes re-injected into the defect⁷⁹. With engineered cartilage, it is difficult to obtain a secure healing of the cartilage to the underlying bone⁷ and there is a danger of dedifferentiation of cells in chondrogenic grafts into fibroblastoid cells⁵.

Tendon and muscle repair with tissue engineering techniques is still at a pre-clinical phase, with promising results in animal trials^8,19. Bone marrow MSC and ADSC were described in several studies as useful in increasing tendon repair and tensile strength¹⁹. Recently was identified Growth Differentiation Factor – 5 as possibly having a significant role in tenogenic differentiation, but further studies are needed⁸⁰.

Engineering of skeletal muscle is challenging due to its complex microelectrical and mechanical networks⁸. Mechanical stimulation, electrical stimulation and vascularization of constructs, with myoblasts or myogenic stem cells in vitro, showed great potential for future approaches^8,81.

PERIPHERAL NERVE DEFECTS

The current replacement options for peripheral nerve defects include autologous, allogeneic and acellular nerve grafts, that are limited by neuroma formation, immune reaction and delayed regeneration, respectively^8,82. Schwann cell transplantation showed capacity to enhance peripheral nerve repair and are considered the most suitable cell type for neural regeneration. However, there is considerable donor-site morbidity, these cells have a slow proliferation rate and cell transplantation alone is limited by suboptimal spatial arrangement^4,8,19. Bone marrow MSC and ADSC have also shown success in enhancing neural regeneration by releasing neurotrophic factors, in vitro and in animal models^8,19,83,84.

VASCULAR TISSUES

When vascular grafts are needed in reconstructive surgery, the actual options are autologous vessels, which can be useless due to a disease, or synthetic grafts, which are prothrombotic⁸. The first bioengineered vessel was clinically used in 1999, on a four-year-old girl, to repair a total occlusion of the right intermediate pulmonary artery⁸⁵. The construct was created by seeding cells extracted from the wall of a peripheral vessel into a biodegradable tube⁸⁵. L’Heureux et al.⁸⁶ created a vessel with cultured human vascular smooth muscle cells without any synthetic material. The vessel displayed similar strength to a human vessel and demonstrated to be functional in vivo in animal models. A clinical trial examined the use of these vessel grafts as arteriovenous shunt in end-stage renal disease patients⁸⁷. Expanded findings from this study showed that the majority of the patients enrolled had successful implantations⁸⁷.

Niklason et al. ⁸⁸ reported a production of arbitrary lengths of bovine vascular conducts from smooth muscle and endothelial cells culture under pulsatile stimuli in vitro; these constructs demonstrated contractile responses including responses to pharmacological agents.

Smooth muscle cells (SMC) have limited proliferation and cultural senescence⁸⁹. One study attempted to derive SMC from human hair follicle stem cells culture. These expressed similar markers to the SMC from human umbilical artery and also showed contractile function⁸⁹. This could be a future reliable source of SMC for blood vessel engineering.

MSCs were also used in vascularization strategies, showing their potential to differentiate into endothelial cells, to promote endothelial repair by secretion of paracrine factors and prevent neointimal formation¹⁹.

Clinical testing on human bone marrow MSCs, ADSCs, fetal stem cells, hematopoietic stem cells and others, showed their vascularization benefits in myocardial post-ischemic neovascularization, neovascularization in systemic sclerosis, myogenic regeneration and neovascularization in erectile dysfunction, angiogenesis in nonrevascularizable limb ischemia, wound neovascularization and cerebral injury revascularization¹⁶.

The cell sheet technique was used as a vascularization strategy, without the use of extrinsic growth factors, with capillary-like structures organization when under hypoxic conditions⁹⁰.

In the lab: To tackle the current limitations related with the vascularization of constructs, the 3B’s Research Group proposed the use of the Stromal Vascular Fraction of Adipose Tissue as a vascularization tool⁹¹. Cell sheets of SVF were shown to restore blood flux in a hind-limb ischemia mouse model⁹⁰. SVF can also be used in combination with scaffolds, allowing their pre-vascularization without the use of growth factors (Figure 3).

CONCLUSION

The potential for tissue engineering is very big, however there are problems still to solve. There is the challenge of meeting the scientific needs while creating a durable and functional tissue and, at the same time, facing the complex regulatory processes until approval for human use³. Researchers need to better understand the role, interactions and fate of stem cells. Large constructs will still be limited by poor blood supply until significantly improved tissue engineering vascularization strategies arise. And the possible side effects (including malignancies) of growth factors recurrent use in tissue engineering is still unknown^3,8,16. Besides these biological problems, there is an increase in health care costs, regulatory restrictions and ethical concerns, that contribute to the delay of tissue engineering widespread application⁸.

To allow this integration of tissue engineered constructs into clinical practice, there is the need for a multidisciplinary team, a clear understanding of manufacturing workflow and to understand the possibility/need of storage into specialized banks^3,8,23.

Tissue engineering has the potential to revolutionize clinical practice as we know it. Small successes so far were achieved, especially in plastic and reconstructive surgery, but there is still a great need for further investigations and clinical trials before this becomes reality.

Statement: There are no conflict of interest in relation with the theme, creating and publication of this manuscript. There was no financial support during the preparation of the article.

Declaration (Ethical Standards): All procedures performed in this study involving human participants were in accordance with ethical standards of the institutional and/or national research committee and with the Helsinki declaration and its later amendments or comparable ethical standards.

Funding: The authors have no financial disclosures to declare.

Conflicts of interest: The authors have no conflicts of interest.

Author(s) Contribution: All the authors have made a significant contribution to this manuscript, have seen and approved the final manuscript, and have agreed to its submission. 

Address for correspondence:

Juliana Ribeiro, MD

Avenida de São Mamede nº 535, 4560-800 Penafiel, Portugal

E-mail: juliana.caalri@hotmail.com