To restore body defects, nature has engineered products since ever, at no costs and introducing no inflammation or rejection side effects. The efficiency of these tissue engineered products is however limited to occur only during the healing process in healthy individuals, and not in compromised patients . While contemplating what nature is doing, human intelligence has been challenged from the prehistoric times to find solutions for filling bone defects, replacing body parts, and healing wounds. Materials such as bone, teeth, wood, metal, ivory and even coral were handled in the aim of restoring body aesthetic and functional integrity. These materials can be considered the first biomaterials ever used; they were used long time before the field of biomaterials emerged in the modern scientific research. In a recent review , Prof. Dorozhkin captures our attention by resuming the most remarkable historical evidences on the existence of biomaterials. "The artificial generation of tissues, organs or even more complex living organisms was throughout the history of mankind a matter of myth and dream. Unfortunately, due to the practice of cremation in many societies, little is known about the prehistoric materials used to replace bones lost to accident or disease. Nevertheless, according to the available literature, introduction of non-biological materials into the human body was noted far back in prehistory." The list of archeological evidences supporting this statement starts with the "Kennewick Man" up to 9000 years ago. With this respect, Prof. Dorozhkin  commented: "This individual, described by archeologists as a tall, healthy, active person, wandered through the region now known as southern Washington with a spear point embedded in his hip. It had apparently healed in and did not significantly impede his activity. This unintended implant illustrates the body's capacity to deal with implanted foreign materials. The spear point has little resemblance to modern biomaterials, but it was a tolerated foreign material implant, just the same" .
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Other similar examples have fascinated the specialists in biomaterials and regenerative medicine; among them there are archaeological testimonies on the innovative spirit of Hindu, Egyptian and Greek civilizations with respect to body parts replacement or repair using interesting techniques or materials. The following examples captivated the attention of high tenure specialists in the biomaterials field and are included in most of the lectures and books on the history of biomaterials:
(1) Wooden prostheses - designed by ancient Egyptian physicians to help their ailing patients. The oldest known prosthesis is a wooden toe fixed with textile lace on the foot of a ~3000 years old female mummy (studied by the German researchers at Ludwig Maximilians University in Munnich) [2-3]. The researchers found evidences on low tissue remodeling after the undoubted toe amputation presumably due to an artery disease . It should be mentioned here that, unless other added fake parts of the body exclusively aimed to provide body integrity for the afterlife journey, this wooden prosthesis presented clear evidence of being used by that woman during her life.
(2) Other biomaterials used by our ancestors include: (i) gold wires and linen thread for ligatures in the repair of bone fractures (apparently used by Hippocrates ca. 460 BC-370 BC), (ii) lint for filling of large cavities, (iii) artificial dental bridges carved from oxen bones by Etruscans, (iv) gold wires used by Phoenicians to bind loose teeth together, (v) dental amalgam to repair decayed teeth (in the year 659 AD) .
(3) A black leg grafted to a white patient - this famous miracle of Sts. Cosmas and Damien was described by multiple paintings and works and it was considered as stating for an early vision on regenerative medicine. Among theses works, the painting "The Healing of Justinian by Saint Cosmas and Saint Damian" by Fra Angelico (ca. 1395 - 1455) is well-known.
(4) Furthermore, the Greek mythology also rings a bell on the regenerative medicine through the idea of regenerating liver in the myth of Prometheus sentenced to eternal suffering "as an eagle ate his liver for eternity while the liver regenerated" .
(5) Impressively, in addition to amputation practiced by Egyptians, trepanation appears as another radical surgical procedure performed by the Incas 500 years ago to treat cranial trauma including intra-cranial pressure. Peru is reported to be the richest area in terms of prehistoric trepanned skulls. However, Verano and Andrushko, anthropologists at Tulane University and Southern Connecticut State University, respectively, reported that little evidence has been found on the assertion that cranioplasty was a common procedure in Peru. Furthermore, the well-known Kanamarca skull is the first "unequivocal case in the Prehispanic Americas where a bone plug is removed and replaced in the trepanation opening" . Another example of old prehistoric trephination procedure is the Crichel Down skull, excavated in England in 1938.
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Regeneration of body parts became even a more critical issue in the modern times we are living, when longevity and life quality are tremendously increasing in connection with the rapid evolution of science and technology. Higher life standards also involved a turnover in the type of pathologies the medicine is facing and, in the last 30 years, more than 60% of all deaths are considered to be due to lifestyle diseases (when compared to influenza, heart disease, pneumonia, and cancer - the main causes of death before 1990's). However, it can be considered that the main success of the second half of the 20th century consists in increasing life expectancy: from 58 years at the beginning of the 20th century to 78 years today. And this statistic is expected to still improve in the near future.
The regeneration capacity of body parts in humans is limited, while most of the "gold standard" therapies remain still the autologous grafts of limited availability and associated to additional morbidity, pain, or even infection at the donor site. In addition to autograft, transplantation is another important and effective long-term treatment for a wide range of health problems. However, recent statistics indicate the reduced availability of donors when compared to the number of suffering patients needing a transplant (i.e. liver donors are available for only 20% of the patients needing liver transplants). Therefore, in the last 20 years, scientists joined their efforts in a multi- and interdisciplinary approach devoted to finding alternatives for transplants.
In this context, one may schematically divide the evolution of implantable materials into four main stages as follows:
The 1st generation of biomaterials (approximately 60 years ago) was represented by already available materials, suitable for implantation. With such materials the success was strictly accidental rather than rationally designed. Examples: gold fillings, bone cements, breast implants, glass eyes, even parachute cloth for vascular implants...
The 2nd generation of biomaterials was designed using common materials with initial non-medical applications. Scientists developed new biomaterials through joint efforts of physicians and engineers. This was the moment when the idea of multidisciplinarity appeared.
The 3rd generation of biomaterials consisted in bioengineered materials developed by the joint effort of biologists, chemists, physicians and engineers. These biomaterials are provided with bioactivity and, very important, with biomimetic properties.
The 4th generation of biomaterials consists in tissue engineered materials. This is the most recent and modern approach. Its development started somewhere about 20 years ago.
Tissue Engineering - Definition and Dimension of the Field.
"When nature finishes producing its own species, man begins, using natural things in harmony with this very nature, to create an infinity of species." This quotation of Leonardo da Vinci was used by Jean-Marie Lehn, a Nobel laureate, when referring to the present and future of Supramolecular Chemistry. In our opinion, from this epitome just two terms were missed - Tissue Engineering.
"Since the early 1990s, the research in biomedical field has been dominated by a paradigm shift whereby the concept of replacing damaged tissues and organs with biomedical devices has been overcome by the goal of their partial or complete regeneration." With this phrase, Professor Matteo Santin (School of Pharmacy and Biomolecular Biosciences from University of Brighton), one of the most respected contemporaneous scientists starts his chapter on high-performance and industrially sustainable tissue engineering, in his book Strategies in Regenerative Medicine edited in 2009 . Further, in this section, different key scientific moments and approaches will be mentioned, in order to briefly refer to the birth of the tissue engineering as a tool to achieve tissue regeneration.
A lot has been told on the first experiments to generate tissues. Generating tissues is equivalent to rebuilding lives and such a pro-life aim deserves all efforts. Approximately 40 years ago, in a pioneering experiment by Dr. W.T. Green, chondrocytes were cultured on bone fragments and the resulting construct was implanted in mice to generate cartilage . But this assumption did not lead to the expected result. However, this experiment was recognized by tissue engineering professionals  as one of the stepping stones of the development in this field; despite being unsuccessful, it allowed a very important and forthcoming vision on the importance played by the cell culture substrate in providing an appropriate environment for the desired cellular events to occur.
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Some years later, tissue engineering officially emerged with the pioneering vision of Langer and Vacanti who announced in Science  that "a new field, tissue engineering, applies the principles of biology and engineering to the development of functional substitutes for damaged tissue" as a response to the fact that "the loss or failure of an organ or tissue is one of the most frequent, devastating, and costly problems in human health care". And this was the first definition ever. The terms Tissue Engineering and Regenerative Medicine are often used with the same meaning. Therefore in many published materials they do appear as Tissue Engineering/Regenerative Medicine. It is however well established that Tissue Engineering develops the biomaterials required by the Regenerative Medicine.
Furthermore, the introduction of both concept and new research field had a strong impact over the scientific community who received this event with huge enthusiasm. Numerous research groups devoted their efforts to this promising field. An indication on the increasing dimension of this research field is given by the fact that more than 46288 scientific research articles were published from 1993 to date. Among them, more than 199 are review articles on different tissue engineering related-aspects, with an interesting subject-evolution (information extracted from PubMed at http://www.ncbi.nlm.nih.gov/pubmed):
1995-2000: bone regeneration
2001-2006: protein- and gene-based tissue engineering, vascular grafts, bone repair, stem cells, growth factors, angiogenesis
2007-2012: bone engineering using genetically modified cells, bone regeneration, growth factors and endothelial cells in therapeutic angiogenesis, nanotechnology, gene therapy, stem cells, osteoblasts in bone tissue engineering, hypoxia inducible factors and mimicking agents in guided bone regeneration, osteogenesis and angiogenesis
Scheme 1. Number of Tissue Engineering Reviews published in the interval 1995-2012, according to PubMed 
Another indication on the development and importance of this research field is that 10 journals are core-devoted to Tissue Engineering: Tissue Engineering, Parts A, B, & C; JournalÂ ofÂ Tissue Engineering, JournalÂ ofÂ Tissue EngineeringÂ and Regenerative Medicine, JournalÂ of Biomaterials andÂ Tissue Engineering, JournalÂ ofÂ TissueÂ Science &Â Engineering, CurrentÂ Tissue Engineering, JournalÂ of Biomimetics, Biomaterials, andÂ Tissue Engineering.
Moreover, medical technology companies appeared into the industrial landscape, having as core or satellite activities the development, manufacturing and marketing of tissue engineered products. Among them: Integra LifeSciences, LifeCell Corporation, Organogenesis, Smith and Nephew, Advanced Tissue Sciences, Genzyme Biosurgery, Cook Biotech, Forticell Bioscience, Zimmer. In 2003 the European Commission identified 113 tissue engineering companies in EU . A recent list with biomaterial-related companies was published .
The increasing interest this field received is also reflected by the fact that Societies for Tissue Engineering were funded over time: (1) TERMIS-EU (formerly European Tissue Engineering Society); (2) The Tissue and Cell Engineering Society (TCES), UK; (3) Korean Tissue Engineering and Regenerative Medicine Society; (4) The Japanese Society for Tissue Engineering, (5) Tissue Engineering and Regenerative Medicine International Society (TERMIS) (2005).
Nowadays, TERMIS covers under its umbrella three regional chapters as follows :
Among other tasks, such organizations are involved in promoting the development of this field in both academia and industry. Another institution activating in this young and powerful research field, the National Tissue Engineering Center (NTEC) was established by the U.S. Congress, in 2002 . All these data state for the explosive evolution of a research field of maximum importance.
In this light, starting with the above mentioned definition given by the parents of the field, and considering the most recent advances, we recall two more recent definitions:
(i) The NIH definition of Tissue Engineering/Regenerative Medicine :
"Tissue engineering / regenerative medicine is an emerging multidisciplinary field involving biology, medicine, and engineering that is likely to revolutionize the ways we improve the health and quality of life for millions of people worldwide by restoring, maintaining, or enhancing tissue and organ function. In addition to having a therapeutic application, where the tissue is either grown in a patient or outside the patient and transplanted, tissue engineering can have diagnostic applications where the tissue is made in vitro and used for testing drug metabolism and uptake, toxicity, and pathogenicity. The foundation of tissue engineering/regenerative medicine for either therapeutic or diagnostic applications is the ability to exploit living cells in a variety of ways. Tissue engineering research includes biomaterials, cells, biomolecules, engineering design aspects, biomechanics, informatics to support tissue engineering and stem cell research."
(ii) The Pittsburgh Tissue Engineering Initiative definition:
"Tissue engineering is the development and manipulation of laboratory-grown molecules, cells, tissues, or organs to replace or support the function of defective or injured body parts. Although cells have been cultured, or grown, outside the body for many years, the possibility of growing complex, three-dimensional tissues - literally replicating the design and function of human tissue - is a recent development. The intricacies of this process require input from many types of scientists, including the problem solving expertise of engineers, hence the name tissue engineering. Tissue engineering crosses numerous medical and technical specialties: cell biologists, molecular biologists, biomaterial engineers, computer-assisted designers, microscopic imaging specialists, robotics engineers, and developers of equipment such as bioreactors, where tissues are grown and nurtured." 
1.2. Tissue Engineering approaches
As a general logo, it can be said that regenerating tissues are rebuilding lives. To succeed in this goal, two tissue engineering approaches were rapidly developed:
in vivo tissue engineering - based on scaffolds able to recruit endogenous cells for tissue repair and
in vitro tissue engineering - using implantable tissue-like constructs obtained from microenvironments generated ex vivo.
Generally speaking, tissue-engineered substitutes fall in one of the two categories:
To understand better the difference between the two classes, we will further refer to skin substitutes. According to the Current Procedural Terminology of the American Medical Association , skin regeneration acellular products such as cadaveric dermis without the cellular material "contain a matrix or scaffold composed of materials such as collagen, hyaluronic acid, and fibronectin". On the other hand, cellular tissue-engineered skin substitutes are products containing living cells such as fibroblasts and keratinocytes within a matrix . The acellular products are easier to bring on the market since they do not contain cells. Good examples in this sense are AlloDermÂ® (LifeCell Corporation) and the two-layer INTEGRAÂ® Dermal Regeneration system.
Furthermore, in an in vitro tissue engineering approach there are several actors that join their efforts in order to ensure the success:
the scientists (i) engineer biomaterial scaffolds able to template and support tissue formation, (ii) cultivate cells into the matrix and (iii) place the construct in a bioreactor to ensure the environment needed for cellular activity;
the cells, the real "tissue engineers", start engineering the tissue in vitro; at a certain development level, the incubation is stopped and the first generation of tissular constructs is obtained;
the surgeon implants the tissular first generation construct into the host, where maturation and integration are expected to occur leading to tissue ingrowth/regeneration .
Once this intimate collaboration between scientists and cells elucidated, it is time to move to another important issue, the applications of tissue engineering products:
* Obtaining functional grafts is the first and most well-known aim of this domain.
* The second field of application is the better understanding of cell behaviour (especially stem cells) in three-dimensional (3D) systems.
* Last but not least, tissue engineered constructs and tissues may be used as model system to understand both physiological as well as pathological phenomena. 
Despite tremendous research supported by both public and private investment, clinical advances in tissue engineering are considered to be still too slow than initially expected and required. Furthermore, the regulatory bodies are responsible for protecting the human health and therefore they are very exigent with respect to the approval of tissue engineered products. A good example in this sense is the fact that FDA (U.S. Food and Drug Administration), the leader in safety regulation, proposed on February 28th 1997 an Approach to the Regulation of Cellular and Tissue-based Products . In this document it was mentioned that "cellular and tissue-based products and their potential uses are too diverse for a single set of regulatory requirements to be appropriate for all. In an effort to develop a comprehensive scheme that would treat like products alike, but that would establish appropriate regulatory distinctions among cellular and tissue-based products in areas where there were differences, the agency identified the principal public health concerns and attendant regulatory issues associated with the use of these products." Among the "Public health and regulatory concerns associated with cellular and tissue-based products" the following are listed: "A) How can the transmission of communicable disease be prevented? B) What processing controls are necessary, e.g., to prevent contamination that could result in an unsafe or ineffective product, and to preserve integrity and function so that products will work as they are intended? C) How can clinical safety and effectiveness be assured? D) What labelling is necessary, and what kind of promotion is permissible, for proper use of the product? E) How can the FDA best monitor and communicate with the cell and tissue industry?" . The document further concludes: "With these concerns in mind, the FDA differentiated cells and tissues and their uses by their risk relative to each concern, so as to enable the agency to provide only that level of oversight relevant to each of the individual areas of concern" . On the other hand, in 2001, the European Commission's Scientific Committee on Medicinal Products and Medical Devices "came to the conclusion that human tissue-engineered products are not appropriately covered by any European regulatory framework" and therefore "A European level regulation was considered essential to guarantee safety and quality of tissue-engineered products applied and traded within Europe or being imported from overseas." .
The Council of the European Communities ensures also a high level of human health and safety, after the last amendment of Council Directives 90/385/EEC relating to active implantable medical devices and 93/42/EEC concerning medical devices for the benefit of patients, consumers and healthcare professionals .
All these emerged in complicated rules the scientists and companies developing tissue engineered materials should respect. However, the fact that tissue engineered products are targeted to save lives makes these concerns reasonable and emphasizes another dimension of the complexity characterizing this novel and extraordinary research field.
1.3. Tissue Engineering Classification
Tissue engineering can be classified using different criteria as described in the following table:
Table 1. Tissue Engineering - classification.
Tissues to be regenerated
Soft Tissue Engineering: tissue-engineered nervous system, tissue-engineered skin substitutes, cardiovascular tissue-engineering, tissue-engineered ligaments and articular cartilage;
Hard Tissue Engineering: Bone tissue engineering
Aim the products are used for
Therapeutic Tissue Engineering
Diagnostic-devoted Tissue Engineering
Use of cells
Cellular Tissue Engineering:
(i) Cells are cultivated on a biomaterial scaffold in a bioreactor before implantation
(ii) Cells and/or bioactive factors are delivered
Acellular Tissue Engineering:
(iii) a bioactive scaffold is used as an instructive environment to in vivo recruit and host cells to regenerate tissues. 
Type of cells
Tissue Engineering based on cells from exogenous sources
Tissue Engineering based on autologous cells
In vitro / In vivo
In vivo approaches: (i) and (ii) above
In vitro tissue engineering: (iii) above 
Tissue-engineered constructs: scaffold with or without cells
Scaffold-free tissue engineering 
1.4. Biomedical applications requiring porous scaffolds
Regenerative medicine needs tissue engineered materials for multiple conventional and unconventional pathologies: trauma, wound healing, ophthalmologic injuries, respiratory and cardiovascular diseases, nervous system, ligament and tendons, hard tissues repair (bone, joints and teeth). Nature provided the human body with extremely complicated and complex structure. For such a brave and challenging aim as generating tissues, scientists should start from understanding the composition, structure, morphology, architecture, regulation mechanisms, physiology and pathology of the target tissue. View the complexity of most tissues, porous scaffolds become crucial for a wide array of applications ranging from hard tissue grafts to soft tissue substitutes. Each of these application fields includes a wide array of sub-classes devoted to the specific needs of the target tissue or organ. Accordingly, soft tissue engineering is concerned with the development of artificial skin, biological substitutes for adipose tissue, for cardiovascular and nervous system, and for artificial lungs and kidneys; hard tissue engineering focuses on bone and teeth regeneration.
Under the form of membranes, sintered or self-assembling particles, bead-based structures, entangled fibrous bodies, meshes, foams, sponges or solid porous blocks, porous matrices should be designed to provide appropriate biomechanical and microarchitectural features in addition to specific chemical structure and bioactivity to optimally assist the cascade of ordered cellular events associated to tissular regeneration.
The porosity is defined as the ratio of void space in a solid and it is a morphological property of a material. The following characteristics are essential in allowing and stimulating cell collonization, tissue ingrowth and, nevertheless, angiogenesis occurrence:
- connectivity of the pores.
In the design of biomaterials, macroporosity is usually characterized by pores superior to 50Â Âµm, while microporosity is associated with pores of approximately 10Â Âµm. A wide range of porosity inducing methods have been developed, ranging from classical techniques such as particulate leaching, particles sintering, assembling fibers, multilayer deposition, thermal treatments (thermally induced phase separation (TIPS) and melt co-continuous polymer blending (MCPB)), to unconventional tools such as laser ablation, computer assisted 3D-printing, electrospinning, or even combinations of the above. However, obtaining scaffolds with predefined porosity remains a challenging task. For example, for bone regeneration, providing an open porosity with pores between 100 to 500 Âµm is essential. On the other hand, the architectural strict requirements should be fulfilled in addition to providing biocompatibility, appropriate stability or degradability, fluid and gas permeability, and, extremely important, mechanical strength for load bearing applications. Furthermore, characterization methods have been developed to qualitatively and quantitatively assess porosity.
Coming back to the development of porous scaffolds for tissue-engineered products, the ever-increasing dimension of this research field can be suggested through mentioning here that an extensive body of literature reports on different aspects related to the importance of porosity for tissues regeneration. A simple search on PubMed using the key words "porous scaffold" generates 2028 articles ; the key words "porous hydroxyapatite" are found in not less than 1860 works , while the terms "porous tissue engineering" are identified in 2913 publications .
2. Tissue engineering constructs
As previously stated, nature provides genetically engineered living bodies with hierarchical architecture up to nanoscale, self-assembling and self-repair ability (musculo-skeletal defects under critical level of damage have the ability to self-repair, while hard tissues constantly remodel).
The current and future trends in the design parameters of biomaterials for tissue engineering are based on composition-structure-properties relationship, moving from the acellular approach to a rational combination of biomimetic scaffolds (polymers, ceramics, metals, composites), biomolecules, cells, and even engineering problems. The scientists developing tissue engineered products have learnt to accept and respect the intimate relationship between function, form and structure. Some key aspects are further discussed.
2.1. Constitutive elements
The definition of tissue engineered products is a structure oriented, as previously stated in the NIH definition of tissue engineering . Accordingly, a tissue engineered construct is built up from the following constitutive elements:
"1) Biomaterials: including novel biomaterials that are designed to direct the organization, growth, and differentiation of cells in the process of forming functional tissue by providing both physical and chemical cues.Â
2) Cells: including enabling methodologies for the proliferation and differentiation of cells, acquiring the appropriate source of cells such as autologous cells, allogeneic cells, xenogeneic cells, stem cells, genetically engineered cells, and immunological manipulation.
3) Biomolecules: including angiogenic factors, growth factors, differentiation factors and bone morphogenetic proteins.
4) Engineering Design Aspects: including 2-D cell expansion, 3-D tissue growth, bioreactors, vascularization, cell and tissue storage and shipping (biological packaging).
5) Biomechanical Aspects of Design: including properties of native tissues, identification of minimum properties required of engineered tissues, mechanical signals regulating engineered tissues, and efficacy and safety of engineered tissues.
6) Informatics to support tissue engineering: gene and protein sequencing, gene expression analysis, protein expression and interaction analysis, quantitative cellular image analysis, quantitative tissue analysis, in silico tissue and cell modeling, digital tissue manufacturing, automated quality assurance systems, data mining tools, and clinical informatics interfaces.
Stem cell research - Includes research that involves stem cells, whether from embryonic, fetal, or adult sources, human and non-human. It should include research in which stem cells are isolated, derived or cultured for purposes such as developing cell or tissue therapies, studying cellular differentiation, research to understand the factors necessary to direct cell specialization to specific pathways, and other developmental studies. It should not include transgenic studies, gene knock-out studies nor the generation of chimeric animals." 
Tissue engineering constructs are complex 3D structures specific to both product type and intended use, designed to be active and to remodel after implantation. They are usually intended to be produced in small lot sizes (even one) and present heterogeneous composition. The construct specification from the in vitro studies is accepted that may not be predictive about clinical safety and efficacy. 
View the scientific background of the authors, further in this work, the interest will be exclusively devoted to the development of scaffolds for tissue engineering applications. The topics will include the selection of the materials and elements of design, in particular the control of biodegradability and porosity. We will share some of our experience in engineering polymer biomaterials for tissue engineering uses.
2.2. Polymer scaffolds for tissue engineering
Since the emergence of Tissue Engineering field, different techniques and materials were used to produce scaffolds: natural, ceramics, polymers, composites or multicomponent complex structures. Each application requires one or another of the above, and the selection is usually based on the correlation between the biological and functional requirements and the composition, structure and characteristics of the material. The interaction of the scaffold with cells, fluids and tissues is strongly dependent on the chemistry of the material (surface and bulk) but also on physico-chemical features that can decisively contribute or affect protein adhesion, cell adherence.
Polymers represent interesting and versatile compounds displaying a large panel of properties (chemical structure, surface properties, bulk physico-mechanical properties and modifications in different environments and as a function of time) that make them suitable for a wide range of tissue engineering applications. These compounds can be processed through various methods to produce films, blocks, fibers or particles with compact or porous architecture. Furthermore, their structural and properties resemblance with the constituents of the extracellular matrix (ECM) renders them useful when addressing scaffold design.
Depending on their origin, polymer molecules fall in one of the categories: (i) natural (i.e. collagen), (ii) semi-synthetic/artificial, (iii) synthetic. Table 2 synthetically presented some of the polymers used to develop tissue engineering scaffolds.
Table 2. Some polymers and their biomedical applications [23-25, 31]
Polymer and combinations
Collagen and Gelatin
wound dressing, gel-like structures, surface or bulk modifications for increased biocompatibility, bone repair, plastic surgery, absorbable sutures, peripheral nerve regeneration, hemostatic agents; drug-delivery systems, cell and drug microencapsulation
Silk fibroin, caseins, albumins
fixation, skin substitutes, bone repair, drug delivery, biophotonics
From vegetable sources
Alginate (marine sources, algae)
impression materials, additive, controlled release of bioactive substances, cells and enzyme immobilization, injectable microcapsules for treating neurodegenerative and hormone-deficiency diseases
Cellulose and derivatives
cell immobilization, drug-delivery systems, dialysis membranes, artificial kidney
solid culture media, immobilization matrix, gel electrophoresis, immunodiffusion, impression material in dentistry
From human and animal sources
Intervertebral disc regeneration, ophthalmology, intra-articular injections, dermal filler, face augmentation, wound repair
Heparin and heparin-like glycosaminoglycanes
antithrombotic and anticoagulant properties
Chitosan and its derivatives
controlled-delivery systems (e.g. gels, membranes, microsheres)
Dextran and its derivatives
plasma expander, drug carrier
modified gelatin or alginate (i.e. with methacrylamide side groups)
PMMA (polymethyl methacrylate) and copolymers
bone cement, bone replacement, dental filler and implants, hard lenses, membranes
PHEMA (polyhydroxyethyl methacrylate) and copolymers
soft contact lenses, skin repair, different resorbable scaffolds, membrane, adhesives catheters, infusion bags, drug delivery, bone cement, joint prosthesis, heart valve, stents, pacemakers, vascular graft, large blood vessels replacement (diameter>6 mm), breast prosthesis
PVC (polyvinyl chloride)
plasmapheresis membranes, blood bags, cardiopulmonary bypass, hemodialysis, intravenous fluid containers, medical tubing
sutures, catheters, plasmapheresis membranes, artificial air duct
PVA (Polyvinyl alcohol)
drug delivery, membranes
cardiovascular applications (large blood vessels (diameter > 6 mm), clips and sutures, coatings, facial prostheses, hydrocephalus shunts, membrane oxygenators
implants in plastic surgery (breast and testicular prostheses), component in skin graft, orthopaedics, blood bags and pacemakers, catheters, drug delivery devices, hydrocephalus shunts, membrane oxygenators
permanently implanted medical devices (prostheses, vascular grafts), catheters, drug delivery systems, artificial heart, ventricular assist devices
Poly(l-lactic acid) (PLA)
Poly(glycolic acid) (PGA)
sutures, drug-delivery systems, tissue engineering, tissue fixation devices (pins, plates, screws)
Pï¥CL (Poly ï¥-caprolactone) and copolymers
bypass grafts, nerve regeneration, flexible material for orthopedics, drug-delivery systems (longâ€term contraceptive device), cell microencapsulation, inert sutures
Poly(trimethylene carbonate) and coâ€polymers with lactides / glycolides
soft tissue regeneration, flexible suture materials, orthopaedic tacks and screws
filler for bone defects, soft tissue repairs
Poly(alkyl cyanoacrylates) and derivatives
surgical adhesives and glues, drug delivery
drug carriers, soft tissue regeneration and engineering, superelastic heart patch, neural
reconstruction applications, endothelialized microvasculature
PET (polyethylene terephtalate)
artificial blood vessel, surgical grafts and sutures
hip joint replacement, hemodialysis, blood oxygenators, blood reservoirs, blood filters, surgical instruments
dialysis membranes, sutures, tissue engineering
PEG (Polyethylene glycol);
PEO (Poly ethylene oxide)
drug delivery systems, excipients, ointment bases, ophthalmic demulcent, organ preservation, encapsulation of islets of Langerhans for treatment of diabetes, biosensor materials
dialysis membranes, antimicrobial polymers
sutures, dressing, haemofiltration membranes
heart valves, penile prostheses, separation membranes
drug delivery devices
tissue culture flasks
Poly(vinyl pyrrolidone) (PVP)
Poly(diol citrates), their copolymers and composites
soft tissue engineering, small diameter blood vessels, gene delivery,
* The mentioned applications do not refer exclusively to clinical use; they include also potential uses, currently under investigation
2.2.1. Selection principles and constraints
The selection of one or another polymer molecule, polymer structure or product synthesis approach depends on various key specifications, further listed:
(i) Mode of application:
(ii) Contact duration:
Limited: < 24 hours;
Prolonged: from 24 hours to 30 days;
Permanent: > 30 days;
(iii) Stability / biodegradability:
Inert or (bio)stable polymer biomaterials. These polymers should be chemically and mechanically stable in biological environments for extended period of time. They are desired for applications requiring long term stability: i.e. heart valve, hip joint;
Biodegradable polymers. Suitable polymers include macromolecules degrading through hydrolytic or enzymatic mechanisms. These materials maintain their function over a predefined limited period of time. Applications include: drug delivery systems and fixation elements: sutures, screws, plates;
(iv) Functionality of the scaffold:
Mechanical role: load-bearing applications, template for tissue regeneration through predefined porosity and specific macro-, micro- and nano-topography;
Therapeutic role: delivery of specific bioactive species.
Furthermore, the choice of the biomaterial is decided using a list of critical technical specifications depending on each application. The list includes without being limited to: biocompatibility, capacity to be sterilized and to be appropriately processed without modification of properties, surface rigidity, specific mechanical behavior, specific topography, economically feasible.
The mechanical properties to be considered are specific to each type of target tissue:
- When the biomaterial is designed as prostheses for hard tissues, the concerns are: modulus (resistance against deformation), fracture strength, resistance to abrasion, fatigue;
- When soft prostheses are to be obtained, properties such as resilience, mechanical strength and fatigue are investigated.
In this context one should be aware that a certain polymer may excellently perform for one application while it can be inappropriate for a different implantable use. Furthermore, the same polymer may be used for totally different applications if it is possible to change its properties accordingly (i.e. PHEMA used both to produce soft lenses as well as bone cements).
2.2.2. Macromolecular components suitable as tissue engineering scaffolds
In this section we will however refer to polymer components we recently investigated for tissue repair/regeneration applications.
The main actors in our research are represented by hydrogels prepared using natural polymers such as collagen, gelatin, sericin, alginate, and synthetic macromolecules such as PHEMA and PAA. These polymers present various characteristics that make them suitable for biomedical applications. Furthermore, their properties can be controlled and modulated to optimally match specific requirements. In an attempt to obtain biomaterials with even more complicated attributes, and overcoming some of the limitations associated to each of the materials as such, we performed a rational combination of two or even three polymers; this strategy lead to multicomponent hydrogels or composites with engineered structure supporting tissue regeneration after cell addition.
It is obvious that knowledge of natural phenomena occurring in the human body gives power to a scientist developing biomaterials for tissue engineering. Thus, it is widely accepted that ECM is responsible for cell adhesion, structural support, active contribution in the occurrence of physiological tissular events, and tissue regeneration under pathological or traumatic conditions. ECM is decisively involved in the tissue physiology since it represents a reservoir of biochemical information; it also acts through specific macromolecular structures and interactions . In this context, for tissue regeneration, our interest went to macromolecular compounds due to the fact that (1) the ECM of soft tissues is "essentially a fiber-reinforced hydrogel composite of structural proteins, polysaccharides, and multi-adhesive glycoproteins" , while (2) the ECM of hard tissues is an organic-mineral composite containing a hydrogel matrix (90% collagen and 10% non-collagenic proteins) reinforced with hydroxyapatite (3Ca3(PO4)2ïƒ-(OH)2) crystals. Generally, the interactions between the ECMs components lead to different elastic gels with specific properties varying from tissue to tissue.
The recapitulation of the chemical, structural and biomechanical environment of cells is essential when aiming tissue engineering . Collagen and gelatin are through their nature suitable for numerous intra-corporeal applications since collagen is the main structural component of ECMs; their biodegradability and thermal sensitivity make them good candidates for one or another uses. Sericin was recently studied for its potential to accelerate proliferation of various cell lines  and moreover, it was showed in experiments in vivo that the silk sericin peptides have no immunogenicity and can be used effectively in biomedical applications .
Furthermore, it is known that polysaccharides are important structural components of ECMs, impairing viscoelastic properties to the hydrogel and resisting compression . In this context, alginate was considered interesting due to its biocompatibility, capacity to generate gels, high hydrophilicity, proved capacity to be cell-loaded. However, it lacks of cell-recognition features and therefore its combination with gelatin is crucial if aiming cellular anchoring onto the scaffold.
The two synthetic polymers mentioned above are extensively researched in various simple or complex formulations due to their high biocompatibility and interesting physico-mechanical properties. In our studies, these materials have been used with the specific task to impart mechanical resistance, to assist the engineering of porosity (i.e. PAA) and to reduce the biodegradability of the proteins (i.e. PHEMA), aspects that will be later emphasized in the next section. From a different point of view, it can be said that the natural products were used to impart biodegradability to the synthetic polymers, and also to provide cellular recognition motifs.
With the main goal of engineering the properties of the scaffolds (biodegradability, biocompatibility, sterilizability, porosity, permeability for nutrients and metabolites, specific mechanical behavior, bio-interaction through adhesion of cells) at low costs and easy processing, our attention went to multicomponent polymer systems allowing to exploit the synergistic effects generated through a rational combinations of polymers. We addressed this aim through the investigation of (i) interpenetrating networks (IPNs) based on gelatin-alginate, gelatin-alginate-PAA and collagen-sericin, collagen-hydroxyapatite, and (ii) polymer networks based on gelatin-PHEMA
3. Biodegradable and Porous Polymer Scaffolds
Biodegradability and porosity are key attributes when engineering biomaterials for tissue regeneration are considered. They impact on the structural and architectural properties, on the in vivo stability and material-tissue interactions, as well as on the capacity of controlled loading and delivery of bioactive molecules of interest. Engineering these two characteristics allows for a rational design of implantable scaffolds with respect to their immediate in vivo performances, but also with respect to the time evolution of the biomaterials upon implantation. The two properties may be ensured individually or in combination. In the latter situation, the synergistic effect should be considered: the biodegradability of the scaffold enhances the porosity in time, while the presence of pores increases the biodegradation rate when compared to compact materials due to easier access of the biodegradation agents into the scaffold.
3.1. Means to provide biodegradability
When biodegradation was initially evidenced, it was considered to be a negative phenomenon leading to a deterioration of properties and performances over time. Nowadays, biodegradation mechanisms are extensively studied in order to be rationally controlled for extremely specific tasks. In this context, most of the drug delivery and tissue regeneration applications request ideal scaffolds with biodegradability occurring over a predicted time interval and with a degradation rate in concert with the tissue regeneration rate. Furthermore, the effect of the biodegradation products is investigated and controlled in order to avoid any undesired effects.
The current definitions of biodegradation as presented in  are: "the breakdown of a material mediated by a biological system" - ESB (European Society for Biomaterials) Consensus Conference II ; or "the alteration undergone by the biomaterial or medical device involving loss of their integrity or performance when exposed to a physiological or simulated environment - ISO ; or "series of processes by which living systems render chemicals less noxious to the environment - Dorland Medical . Biodeterioration is further defined as "the process of change in characteristics of a substance, material or object that arise from its presence in a biological environment and which cause an undesirable reduction in overall quality" .
The easiest way to obtain biodegradable polymer biomaterials is to start from macromolecules that are able to biodegrade under the specific conditions of each application. In this aim, one may select available or easy to prepare natural or synthetic polymers such as: collagen, gelatin, alginate, chitosan, hyaluronan, fibrin, polyhydroxyalkanoates such as poly(propylene fumarate) (PPF), poly(hydroxybutyrate) (PHB), polyesters such as PLA, PGA and their copolymers, PCL, polyorthoesters, poly(dioxanone), poly(anhydrides), and their combinations.
The factors known to influence the degradation of polymers are (without being limited to):
(i) material-based: the chemical structure and the chemical composition, the distribution of structural units in multimers, the polydispersity and the molecular weight, the functionality and the presence of chain defects, the morphology (microstructure or nanostructure if any, crystalline phase, phase separation);
(ii) processing and storage - based: processing methods, method of sterilization, storage history, implantation site and in vivo interactions including protein absorption and enzymatic attack, physiochemical characteristics of the product (shape, size), degradative mechanisms.
Furthermore, the occurrence and the intensity of biodegradation phenomena are enhanced by a series of factors including increased hydrophilicity of the polymer chains (in terms of backbone or/and endgroups), low level or absence of crystalline phase, enhanced porosity (eventually interconnected), small dimension of the product/device.
However, some of the biodegradable materials, despite biodegradability and biocompatibility do not possess appropriate mechanical behavior or the biodegradation rate needs to be improved or better controlled. On the other hand, a lot of interesting biomaterials do not exhibit biodegradability, this limiting their application in tissue regeneration. Therefore, strategies to modulate different specific properties including biodegradability were developed.
I. The influence of biodegradable components.Â The combination of biodegradable and non-biodegradable components is extremely investigated. With this respect we recently prepared multicomponent scaffolds based on semi- and inter-penetrating polymer networks based on collagen-sericin , gelatin-alginate, gelatin-alginate-PAA , gelatin-PHEMA . The guiding idea was that collagen, gelatin, and even alginate will impair biodegradability to the resulting bi- or tricomponent scaffolds while presenting improved overall properties. To be more specific, the presence of gelatin in the scaffold gelatin-alginate-PAA ensures enzymatic biodegradation of the polymer substrate. Without the addition of gelatin, PAA and alginate would not have been enzymatically attacked in the in vivo environment. We studied these aspects through the in vitro collagenase digestion of multicomponent hydrogels. The effect of biodegradable compounds was experimentally confirmed. Figure 1 is representative with this respect. Figure 1 presents the FTIR confirmation of enzymatic degradation as recorded for a sample of gelatin-alginate-PAA with a content of gelatin of 52%. Briefly, the materials were submitted to collagenase treatment and FTIR spectra were recorded before and after the enzymatic attack. Gelatin and PAA were used as control. After the enzyme treatment, the FTIR spectrum of the analyzed sample confirms a decrease of gelatin content in the remaining material. This is visible in the two wavenumber intervals indicated as (i) and (ii) on the figure. The first evidence is considered to be the modification of the broad peak observed above 3000 cm-1. The intensity of this peak is increasing with the consumption of the protein by the enzyme, while the shape of the peak is maintained. Furthermore, C-H vibrations at 2840-2940 cm-1 are more intense with decreasing gelatin in the matrix, result explained through stronger influence of PAA and alginate in the remaining polymer. The second area of interest is denoted (ii) and it mainly offers information on the amides-characteristic vibrations. Gelatin consumption after the enzymatic treatment is proved by a strong modification of the signals associated to the (ii) area: the two peaks at approximately 1650 and 1612Â cm-1, characteristic to overlapping C=O from alginate, amide I, amide II, and C=O from PAA, become three peaks due to reduction of amide I and amide II from gelatin, simultaneously with more intense C=O from alginate and amide from PAA.
Fig.1. FTIR spectra emphasizing the enzymatic degradation of gelatin from gelatin-alginate-PAA hydrogels when compared to control hydrogels gelatin and PAA; for gelatin-alginate-PAA brown line - gelatin, blue line - tricomponent scaffold before collagenase treatment, and green line - tricomponent scaffold after enzyme treatment. i - spectral modification of the stretching vibration characteristic to O-H and N-H; ii - spectral modification of the amide I and amide II vibrations following gelatin enzymatic digestion.
Correlations between the composition of the multicomponent systems and the biodegradation level were also established. As a general rule, increased amount of biodegradable component is associated with higher biodegradation extent of the multicomponent scaffolds. Figure 2 offers an example for such behavior, for a series of three compositions gelatin-alginate-PAA.
Fig.2. Enzymatic degradation extent of multicomponent gelatin-alginate-PAA hydrogels with different compositions. Decreasing the amount of gelatin in the hydrogel is increasing the scaffold stability in terms of resistance to enzymatic attack.
In the case of covalently combined gelatin-PHEMA systems, it was demonstrated that the presence of the synthetic component decreases the degradation degree of the scaffold; from the opposite point of view, the use of a biodegradable component, gelatin, leads to partially biodegradable materials based on PHEMA. Increasing the HEMA content in the initial reaction mixtures is associated to lower biodegradation level of the final materials . Thus, the bicomponent scaffolds prepared when using 33% HEMA in the polymerization mixture degraded to a total degradation extent of 69% after in vitro collagenase digestion. The degradation extent was successfully controlled through the balance between the natural and synthetic components of the bicomponent materials, generating scaffolds with fully controlled degradation behavior .
II. The influence of the water affinity of the scaffold. Besides the decisive influence of the addition of biodegradable components into a complex scaffold, other factors allow for a superior control of the biodegradability. Among them, as stated before, the water-affinity plays a very important role. In our research on the rational design of multicomponent scaffolds with tailored properties, great attention was focused on the influence of extremely hydrophilic partners onto the biodegradation rate. We established that incorporation of macromolecular elements with high water affinity such as PAA and alginate enhances the degradation rate of multicomponent scaffolds due to improved accessibility of the substrate to hydrolytic attack [30, 31].
III. The influence of crosslinking. Another key parameter in the occurrence of biodegradation is the crosslinking. This has impact on the stability of the scaffolds through: (1) the nature of the crosslinker, its dimension, stability, chemical structure and (2) the crosslinking degree. Augmenting the crosslinking density reduces the biodegradation rate since it leads to a denser hydrogel network.
These are only few examples of biodegradation influence. Their rational combination allows for a superior control of the biodegradation in order to optimally suit specific tissue regeneration applications.
3.2. Means to provide porosity
Porosity. As stated before, this is a morphological property independent on the composition of the material. For a wide range of tissue engineering applications porosity is crucial. Therefore, the methods to generate porous scaffolds, the means to control their morphology and the porosity assessment techniques are key issues that should be addressed by the materials science specialists.
The methods of generating porosity were briefly listed in section 1.4. These methods provide porous constructs starting from polymers or from monomers, via simple or more complicated processes. Porous matrices may be obtained using:
(i)Â solid bulk materials - supercritical fluids technology, laser ablation, 3D-printing;
fibers, or particles - (self-)assembling, sintering;
polymer solutions - electrospinning, rapid prototyping, 3D-printing, thermal induced methods such as freeze-drying;
polymerization mixtures - foam forming methods.
The selection of one or another technique starts from the analysis of the biological application and of the polymer material; the decision is limited by a number of practical considerations such as: the required physico-chemical and mechanical properties, available devices and infrastructure, toxicity of the method, and, nevertheless, associated costs. For instance, it is widely recognized that particulate leaching is easy to perform but can be associated with defects due to incomplete removal of the porogen, while 3D-printing is a modern technology generating 3D-products with controlled porosity in terms of size, distribution and orientation, morphology, and connectivity.
Means to control porosity. A number of strategies were developed in order to control the porosity of scaffolds in terms of predefined even hierarchically organized morphology and connectivity level [32, 33].
Various geometrical models for particles and fibers assembling have been established and investigated; this is useful both for directly obtaining porous bodies with pores between the particles/fibers, as well as for specific template with effect on void formation after template removal/dissolution.
a. Methods involving polymerization reactions:
In addition to a simple variation of the polymerization conditions, tailoring of porosity can be carried out by varying several factors including the monomer composition, the type of initiator, the use of solvents in the polymerization mixtures, the use of porogen species or foaming agents, crosslinker type and crosslinking degree.
b. Methods not involving polymerization reactions:
These methods refer to the use of polymers as such (solutions, particles, fibers) to create porous bodies with predefined architecture in terms of size and shape of the pores, interconnectivity, and porosity level.
Fig.3. Porous bodies with pores obtained through the entanglement of polymer fibers (unpublished results)
Various techniques are developed in this aim as already emphasized above. Further in this section, we will only present few practical examples on how porosity can be controlled via rational combination of strategies.
I.Â Composition adjustments. Various modifications of the composition may be performed in order to better suit the generation of pores using a selected method. A relevant example with this respect consists in the increase of the water-affinity of the scaffold when generating porosity through freeze-drying. Freeze-drying (lyophilization) is a well known method for the fabrication of porous biomaterials based on natural polymers, being able to preserve their biological activity and stability. The process is performed in three steps: freezing, main freeze-drying and final freeze-drying. The porous structure is formed as a result of ice crystal formation during freezing of a polymeric suspension, leaving voids that generate pores [34-36]. During freeze-drying, the ice is removed by sublimation and the empty pore remain in the place of every ice crystal.
As examples of composition adjustment that affect the porosity of the scaffold, we recently prepared various bicomponent hydrogels including collagen-sericin, gelatin-PHEMA, gelatin-alginate. Ä°t is well known that collagen scaffolds produced by freeze-drying present open interconnecting porous networks . The use of a second component modifies the morphology of the resulting porous hydrogels with respect to the size, shape, and interconnectivity of the pores . The same influence was observed when gelatin is covalently combined with PHEMA (methodology and compositional details described in ). In this respect, the use of increasing amount of HEMA in the polymerization mixtures leads to important changes with respect to the porosity of the gelatin scaffolds used as control. Hence, in figure 4 two distinct morphologies are shown: longitudinal channels with top-to-bottom orientation are characteristic for freeze-dried gelatin hydrogels, while the use of the synthetic component leads to smaller pores, with round and ovoidal shape and higher degree of interconnection. Further compositional adjustment allowed for the corelation of the architectural features with the gelatin : PHEMA ratio .
Fig. 4. Morphological changes with respect to the porosity of gelatin hydrogels and gelatin-PHEMA hydrogels.
II. Control over the fabrication parameters/ conditions
The parameters used to fabricate porous materials are extremely important when aiming porosity control. Various applications may request random or predefined porosity and the polymers should be engineered with respect to each specific need. After a method for porosity generation is selected, the processing control is the key factor in obtaining the desired microarchitecture in terms of porosity. To better explain this, we will use again the example of the freeze-drying of natural polymer aqueous solutions.
The freezing temperature and freezing rate are the most important factors which influence the porosity, pore size and pore morphology of freeze-dried collagen-based scaffolds . According with Gelinsky et colab.  the freezing process at temperatures between -20 and -250C leads to an interconnecting porous systems with pore diameter of aproximately 200 Âµm. To obtain smaller diameter of pores it requests freezing at much lower temperatures.
Furthermore, O'Brien et colab.  showed that collagen-glycosaminoglycan scaffolds processed by freeze-drying at freezing temperatures of -40, -30, -20 and -100C exhibit a mean pore sizes of 95.9, 109.5, 121.0 and 150.5 Âµm respectively. The results of our previously studies showed the influence of the freezing temperature on the mean pore sizes for some cross-linked collagen-doxycycline matrices . The collagen-doxycycline scaffolds presented a porous structure with a large variation in average pore diameter, from 50 to 250 Âµm depending on the freezing temperature.
Modifications to the freeze-drying process have allowed a large range of collagen scaffolds with mean pore sizes ranging between 85 and 325 Âµm .
The pore diameter in spongious structures obtained by lyophilization can be controlled by other factors such as: concentration of collagen in suspension, pH of the suspension, composition of suspension. The results presented in the literature showed that the higher collagen concentration, the small pore sizes and the decrease of pH produces small pores in collagen fibers [43, 44].
The composition of collagen-based scaffolds influences the pore sizes, as indicated in Figure 5.
Fig.5. SEM images of collagen-based scaffolds: a) Collagen, x400; b) Collagen-Sericin (100:40), x 400; c) Collagen-hydroxyapatite (100:30), x 200; d) Collagen-hydroxyapatite (100:30), x 2000.
All the studied collagen-based scaffolds processed through freeze-drying showed highly porous networks, with two types of internal architectures . The control sample, consisting in collagen, shows fibrillar structure with inner like structure of 92÷150 Âµm diameter as it can be seen in the Fig. 5a. The scaffold based o