Regenerative medicine: Historical roots and potential strategies in modern medicine.

Regenerative medicine is a distinct major advancement in medical treatment which is based on the principles of stem cell technology and tissue engineering in order to replace or regenerate human tissues and organs and restore their functions. After many years of basic research, this approach is beginning to represent a valuable treatment option for acute injuries, chronic diseases and congenital malformations. Nevertheless, it is a little known field of research. The purpose of this review is to convey the state of the art in regenerative medicine in terms of historical steps, used strategies and pressing problems to solve in the future. This review represents a good starting point for more in-depth studies and personal research projects.

1. Historical background

Regenerative medicine (RM) implies the replacement or regeneration of human cells, tissue or organs, to restore or establish normal function [1].

The term “regenerative medicine” is widely considered to be coined by William Haseltine during a 1999 conference on Lake Como, in the attempt to describe an emerging field, which blent knowledge deriving from different subjects: tissue engineering (TE), cell transplantation, stem cell biology, biomechanics prosthetics, nanotechnology, biochemistry [2]. Historically, this term was found for the first time in a 1992 paper by Leland Kaiser, who listed the technologies which would impact the future of hospitals [3].

RM is considered a novel frontier of medical research, but the idea of creating artificial organs is not so recent. In 1938, a book, called “The culture of new organs”, was published and the authors were Alexis Carrell, a Nobel Prize winner for his study on vascular anastomosis, and Charles Lindbergh, the first aviator to fly across the Atlantic alone. Wondering why his sister-in-law's fatal heart condition could not be repaired surgically, Lindbergh, despite not being a professional, ended up working together with Carrell at Rockefeller Institute for Medical Research during the 1930s, where they created an artificial perfusion pump allowing the perfusion of the organs outside the body during surgery: their work was the basis for the development of the artificial heart [4].

The regeneration of body parts is a rather common phenomenon in nature; a salamander can regenerate an amputated limb in several days [5]. Humans have this ability as well, but they lose it over the years: a severed fingertip can regenerate until 11 years of age [6]. The human regeneration potential was well-known also in ancient times, as demonstrated by the myth of Prometheus: his liver was eaten by an eagle during the day and it completely regenerated itself overnight.

During the last centuries medicine has gained many successes: antibiotic therapy, anesthesia, sterilization, etc. However, there are still many pathologies which cannot be treated by preserving the affected organs, but require the resection of lesions or the repair with autologous tissues or even the replacement with allografts [7]. This is the three R's paradigm in traditional surgery with three solutions, all of which pose different problems.

When a surgeon resects an extensive part of small bowel, leading to a malabsorption syndrome, called short bowel syndrome, long-life total parenteral nutrition is imposed, threatening patient's life [8].

People suffering from high-pressure or poorly compliant bladders may need augmentation cystoplasty, which is performed by using part of small bowel. Since gastrointestinal tissues adsorb solutes rather than excrete urine, the repaired bladder is often complicated by increased mucous production, infections, metabolic disturbances, urolithiasis, perforation and even cancer [9].

As for organ replacements, in 1954, the kidney was the first organ to be substituted in a human, but between identical twins so that rejection did not arise [10]. Later, also cell transplantation was achieved: an immunodeficient patient received his sibling's bone marrow [11]. At first, transplants were relegated to research because of the adverse immunological responses, but the advent of cyclosporine in the 1980s transformed transplantations into life-saving treatments, as the risk of rejection could be drastically reduced. Nowadays, lifelong immunosuppression carries many side effects, representing one of the two big problems related to transplantations [12]. The other one is the shortage of donors, not being able to meet the ever increasing demand of organs [13]. Due to the progressively aging population, transplantations will be increasingly needed to replace end-stage diseased organs injured by age-related diseases.

All these issues are carrying with them economical and social problems: while in 1941 there were 41 workers for one retiree in the USA, now there are only three workers for one retiree, so that the common invalidating chronic diseases are bearing upon a small part of work-age citizens [14].

Therefore, medicine is facing with pressing problemswhich require an evolution of medical treatments and theregeneration of damaged tissues, “the fourth R”, could revo-lutionize modern medicine, offering the way to cure, ratherthan merely treat symptoms.

A partial list of the most relevant conquests in the his-tory of regenerative medicine is reported in Table 1.

Table 1

A partial list of firsts in RM.

Year	First
1968	First cell transplantation: bone marrow transplant [11]
1978	Discovery of stem cells in human cord blood [15]
1981	First in vitro stem cell line developed from mice [16]
1981	First engineered tissue transplantation: skin [17]
1996	Creation of the first cloned animal: a sheep, named Dolly [18]
1998	Isolation of human embryonic stem cells [19]
1999	First laboratory-grown organ: an artificial bladder implanted in a patient suffering from myelomengicocele [20]
2004	Implantation of first engineered tubular organs (urine conduits) [21]
2007	Discovery of stem cells derived from amniotic fluid and placenta [22]
2009	First solid organ engineered by recycling donor liver [23]

The later part of the article looks into the current strategies used in RM and the associated shortcomings whichdeter its liberal and convenient application in the clinicalsetting on a daily basis.

2. Current strategies used in regenerative medicine

There are substantially three different approaches topursue the objective of RM (Fig. 1):

Fig. 1

Strategies used in regenerative medicine. There are substantially three approaches: cell-based therapy, use of engineered scaffolds and the implantation of scaffolds seeded with cells.

Cell-based therapy;

Use of either biological or synthetic materials to leadrepair processes and cell growth;

Implantation of scaffolds seeded with cells.

2.1. Cell-based therapy

Humans have complex multicellular framework withseveral types of cells specialized in particular functions. However, all cells descend from one unique cell, calledzygote. During development, cells differentiate progres-sively and acquire more and more specific tasks, while theylose their capacity for differentiating into other cells. Theability to differentiate into other cell types is defined as”cell potency” (Table 2).

Table 2

Cell potency.

Totipotency	The ability of a single cell to produce all cells (potency possessed until 16-cell stage during blastocyst phase)
Pluripotency	The ability to differentiate into a cell of all three germ layers (e.g. embryonic stem cells)
Multipotency	Gene activation limits these cells to differentiate into multiple, but limited cell types (e.g. hematopoietic stem cells can differentiate into all blood cells: erythrocytes, lymphoid cells, neutrophils, platelets, etc.)
Oligopotency	The ability to differentiate into limited cell types (e.g. lymphoid stem cells become either B cells or T cells)
Unipotency	Ability to differentiate into one single cell type (e.g. precursor cell)

Cell therapy consists of injecting novel and healthy cellsin pathologic tissues. It can rely either on already differen-tiated cells or on undifferentiated stem cells (Fig. 2), whichcan differentiate depending on particular circumstances.

Fig. 2

Cell therapy bases on the injection of cells obtained by different methods. Adult primary cells are taken from patient and directly implanted after expansion in vitro. Biopsied tissues contain adult stem cells (ASCs) to expand, differentiate into a specific type and implant. Adult skin cells may be reprogrammed through specific transcription factors in order to obtain induced pluripotent stem cells. Embryonic stem cells are derived from the inner cell mass of a blastocyst. At last, the amniotic fluid is a potential source for stem cells (AFSCs). Read the text for a detailed description. Abbreviations: AFSCs, amniotic fluid-derived stem cells; ASC, adult stem cell.

On the first hand, the differentiated endogenous pri-mary cells are collected by patient's specific tissues with the advantage of being ready to implant without any fur-ther manipulations, but expansion. However, it is difficultto get a considerable number of these cells in vitro, also for organs (e.g. liver) with a great replication potential invivo, as the cells lose the usual microenvironment neededto proliferate [24]. Therefore, these cells will be used lessand less in the future, even if they are not correlated withrejection and important inflammatory responses.

On the other hand, stem cells (SCs) can proliferateextensively, with the capacity of self-renewing whilethey maintain their undifferentiated state, until they areinduced to differentiate into a specific cell type [25]. SCs canbe obtained in several ways. They are autologous if derivedfrom patient, allogeneic if derived from a human donor andxenogeneic if derived from another animal.

Adult stem cells (ASCs) had been isolated from nearly allhuman adult body tissues, where their goal is to restoreoriginal tissue function after minor injuries [26]. Amongthese cells, a preeminent role is played by the bonemarrow-derived mesenchymal stem cells (MSCs), as theyhad been studied deeply. Using different culture protocols, they have been shown to be able to differentiate into manykinds of cells, useful to treat bone, cartilage, nervous, mus-cle, cardiovascular, blood, gastrointestinal diseases [27].

By aspirating the inner cell mass from an embryo during the blastocyst stage (5 days post fertilization), we can get embryonic stem cells (ESCs), which can proliferate exten-sively while maintaining their pluripotent state until theyare induced to differentiate into one kind of cells from all the three embryogenic germ layers (Table 3) [28]. Human ESCs can be derived from the surplus of embryos gener-ated during in vitro fertilization. Besides the huge potential, there are several issues with the use of ESCs which cannot be ignored:

Table 3

Derivates from germ layers.

Germ layer	Derived tissues and organs
Ectoderm	Epidermal tissues and nervous system
Mesoderm	Bone, blood, cartilage, muscle, urogenital system, serous membranes
Endoderm	GI tract, airways

- Rejection: they are allogeneic, but immune responses can be avoided with some new developing technologies, like therapeutic cloning and adult cell reprogramming;

- Need for feeder cells for trophic support: first time feeder cells were used in mouse fibroblasts, correlated with the risk of xeno-contamination [29];

- Carcinogenesis: ESCs can develop into teratomas [30];

- Ethical and moral issues: they represent a notable source of debate, as their source is an embryo, whose development is interrupted by the aspiration.

The first recipient of these cells was a young man, who had a spinal cord injury in a car accident: he received the injection of oligodendrocytes obtained from ESCs [31].

Another strategy to obtain ESCs is the so-called therapeutic cloning or somatic cell nuclear transfer (SCNT), which is based on the transfer of a somatic cell nucleus into an oocyte. In this way, early stage embryos are cultured to produce ESCs with the potential to become almost any adult cell types [32]. Besides being a source for RM, this technique is also the basis for cloning animals, like the famous sheep, called Dolly [18]. Through this technology, pathologic cell lines can be obtained to study the effects of some molecules in cells with specific diseases.

Autologous stem cells can also be obtained through a reprogramming of adult cells to get induced pluripotent stem cells (iPSCs). They have the same cell potency as ESCs, so they could replace the controversial use of ESCs. The generation of iPSCs was firstly achieved by introducing a series of transcription factors into murine fibroblasts: the firsts were OCT3/4, SOX2, KLF4 and c-MYC [33]. The latter, c-MYC, is an oncogene and could give rise to tumors, so it had been replaced successfully, according to different reports, using OCT4, SOX2, NANOG and LIN28 [34]. Unfortunately, in this way the process took longer and was not as efficient as the other protocol. Originally, the delivery of these transcription factors was achieved through the use of retro- and lentiviral constructs, but, since this strategy could provoke insertional mutagenesis and oncogene activation, it should be substituted by non-viral-based methods or by the adenovirus-based transient transfection without genomic integration [35]. Recently, iPSCs were obtained in mice in vivo without the use of a Petri's dish [36].

Another important feature of iPSCs is their use for the generation of disease-specific lines (for example, affected by Parkinson's disease, Alzheimer's disease, diabetes mellitus type I) to study disease mechanisms and drug screening [37].

A human clinical trial with iPSCs is being conducted at Japanese RIKEN Center for Developmental Biology [38]. The first recipient was a 70-year-old woman affected by exudative age-related macular degeneration (AMD), whose skin cells were taken and induced to differentiate into retinal pigment epithelium (RPE) cells, which were used to create a small monolayered RPE sheet to implant into the patient's eye, without any biomaterials.

Lastly, scientists can obtain SCs from amniotic fluid and placenta by amniocentesis or chorionic villous sampling in the developing fetus or from the placenta at birth, the so-called amniotic fluid-derived stem cells (AFSCs) [22]. These cells are multipotent and do not develop neoplasms. A range of possible clinical applications has been described in the literature. For example, AFSCs from cell banks can represent a lifelong autologous source for heart-valve replacements [39]. Since they are very promising, they have been studied deeply in the recent past, but no human clinical trials have been performed yet.

All the advantages and disadvantages related to the different cell types are summarized in Table 4.

Table 4

Advantages and disadvantages of cell types used in RM.

Cell type	Advantages	Disadvantages
Differentiated endogenous primary cells	No tissue rejectionReduced inflammatory response	Difficult expansion because of in vitro short lifespanDifficulty in getting healthy cells in diseased organs
Adult stem cells (ASCs)	No tissue rejectionNo ethical problemsNo tumorsEasy isolationIn some cases easy access (e.g. apheresis and subcutaneous fat)	Low number in each tissueDifficult in vitro expansion without differentiation
Embryonic stem cells (ESCs)	Unlimited ability to self-renewPotential to differentiate into many specialized cells from all the three germ layers	Ethical and political problemsTumorigenityNeed for feeder cell layers (risk of xeno-contamination when mouse fibroblasts are used)
Induced pluripotent stem cells (iPSCs)	Similar as ESCsEasier generation than ESCsNo ethical problems	Tumorigenity
Amniotic fluid-derived stem cells (AFSCs)	Great ability to proliferate without feeder cellsNo tumorigenicNo ethical problemsPossibility of preservation as lifelong autologous stem cells together with other perinatal stem cells (umbilical cord placenta and amnion membrane-derived stem cells)Possibility of ante-natal collection by amniocentesis or chorionic villous sampling	Further research is needed (being the latest discovery)

2.2. Biomaterials

Tissues generally consist of cells and extracellular matrix (ECM). Biomaterials usually serve as ECM, giving both structural and functional support. During the last few years, ECM has been shown to play a key role in many different functions, such as gene expression, survival, death, proliferation, migration, differentiation. Therefore, all of them should be reproduced by biomaterials enriched with bioactive factors, such as growth factors and cytokines. The materials used in RM (Table 5) can be classified as natural or synthetic with different advantages and disadvantages.

Table 5

Examples of biomaterials used in RM.

Origin	Examples
Natural materials	Collagen, fibrin, chitosan, dextran, alginate, gelatin, cellulose, hyaluronic acid (HA), silk fibroin
Acellular tissue matrix	Bladder acellular matrix (BAM), small intestinal submucosa (SIS), bowel acellular tissue matrix (ATM), bovine pericardium (BPV), human placental membrane (HPM)
Synthetic polymers	Polyglycolic acid (PGA), polylactic acid (PLA), poly(lactic-co-glycolic) acid (PLGA), polycaprolactone (PCL), poly(copralactone-co-ethyl ethylene posphate) (PCLEEP), polydioxane (PDS), polyethylene glycol (PGE), poly-N-(2-hydroxyethyl)metacrylamide (PHEMA), poly-N-(2-hydroxypropyl)methacrylamide (PHPMA)

Synthetic materials can be identically reproduced on a large scale with specific properties of microstructure and degradation rate. However, they have the important drawback of lacking of biologic recognition. Nevertheless, different research groups are trying to solve this problem by incorporating the molecules to help the recognition of synthetic scaffolds. By assembling electrospun nanofibers and self-assembling peptides with functional motifs (Fig. 3), Gelain et al. created neural prosthetics which had shown to lead nerve regeneration in rats with chronic spinal cord injury [40].

Fig. 3

Schematic models of the self-assembling peptide used by Gelain et al., RADA16 (blue bars), extended though several different functional motifs (different colored bars) in order to design different peptides. A schematic model of a self-assembling nanofiber scaffold with combinatorial motifs carrying different biological functions is shown right.

Source: Gelain F, Bottai D, Vescovi A, Zhang S. Designer Self-Assembling Peptide Nanofiber Scaffolds for Adult Mouse Neural Stem Cell 3-Dimensional Cultures. PLoS One. 2006 Dec 27;1:e119

On the other hand, natural materials can be integrated perfectly. They can be furnished by other living organisms, but in these cases they present cellular components which may induce an immune response. The latter can be avoided by making use of detergents (e.g. trypsin/TritonX-100) which leave only ECM, creating the so-called acellular tissue matrices [41]. Unlike synthetic ones, both naturally derived materials and acellular tissue matrices can not be produced easily in large quantities according to good manufacturing practice.

The most used natural biomaterial is probably the hyaluronic acid, which is a common anti-aging product in skin-care products and injectable facial fillers. As for sample acellular tissue matrix applications, Portis et al. demonstrated the feasibility of laparoscopic bladder augmentation in minipigs using porcine bowel acellular tissue matrix and porcine small intestinal submucosa [42].

The ideal biomaterial should be biocompatible and biodegradable at the same rate as regeneration process without leaving toxic end-products, interfering with regeneration process and causing inflammation and/or obstruction [43].

Another important feature is porosity, which allows the exchange of nutrients and wastes. This property is extremely difficult to be achieved successfully and 3D bioprinters seem to be the perfect solution of this problem. The research group guided by Shaochen Chen bioprinted a 3-D liver-like device to detoxify blood, by encapsulating functional nanoparticles in a biocompatible hydrogel [44]. Thanks to a technology, called dynamic optical projection stereolithography, complex 3D microstructures, like blood vessels, can be printed within few seconds. Without vasculature printing, essential for distributing nutrients and oxygen, tissue-engineered organs, such as liver or kidney, are useless in clinical practice. The biofabrication technique grounds on a photo-induced solidification process, which uses soft biocompatible hydrogels containing living cells and forms one layer of solid structure at a time, but in a continuous fashion, by shining light on a selected area of a solution containing photo-sensitive biopolymers and cells [45]. Other current 3D biofabrication techniques, such as two-photon photopolymerization, can take hours to fabricate a 3D part. At last, Organovo is a medical start-up intending to deliver bioprinted organs, like liver, for surgical therapy and transplantation [46].

2.3. Implantation of scaffolds seeded with cells

This approach is a combination of the previous two strategies.

In 2006, Atala et al. reported autologous engineered bladder constructs could be used in patients suffering from myelomeningocele needing augmentation cystoplasty [20]. The synthetic scaffold was made up of collagen and PGA and seeded with patient's urothelial and smooth muscle cells, respectively on the endoluminal and abluminal side. These cells were obtained through a patient's biopsy and expanded in vitro before scaffold seeding.

Another example is the realization of a bioartificial liver obtained through the decellularization process [23]. The latter consists of eliminating all liver cells preserving the structural and functional characteristics of the vascular network, which allows the organ perfusion. Later, adult hepatocytes recolonize liver matrix and support physiological functions, like albumin excretion and urea synthesis. The liver grafts obtained in this way were successfully transplanted into mice, paving the way for a new approach to the treatment of end-stage liver diseases.

3. Conclusions

RM opened new avenues for curing patients with difficult-to-treat diseases and physically impaired tissues. Despite many successes, RM is still unfamiliar to many scientists and clinicians. This poses a great limit, as tissue engineering and regenerative medicine could overcome the unsolvable problems of the current medical treatments.

In order to resort to RM in the clinical setting on a daily basis, it is mandatory to obtain important financial investments from different sources including governments and industries that are oriented toward research and medical innovation. There is a considerable need for long-term vision and support for RM to accelerate the development of novel therapies and to promote the stability of collaborations around the world.

In addition to the financial and technical concerns, process development is a significant hurdle to manage with and it includes intellectual property, manufacturing, and logistical concerns. Cell therapy and tissue engineering have the potential to revolutionize patients’ care. But in order to materialize this concept, ideas generated in the laboratory need to be taken through process development and transformed into widespread commercial products. Licensing, legal support, logistics, supervision at the governmental level, unexpected failures, and team management play a pivotal role.

Since RM is a cross-sectional area of research, a multidisciplinary team, including doctors, biologists, bioengineers, chemists, and surgeons, is required to initiate and master the key steps involved in cell therapy and tissue engineering. This necessitates the need for training courses of cell culture, stem cell technology, tissue engineering and experimental surgery.

The crucial point of this revolution is transforming the current numerous scientific discoveries into novel and viable therapies: from bench to bedside.

Conflict of interest

None declared.