Prolotherapy is defined as a nonsurgical regenerative injection technique that involves injecting sclerosing or proliferant substances into painful tendon insertion sites (entheses), ligaments, joints and adjacent joint spaces in single or several injection sessions to induce normal tissue growth in injured areas [1] [2] .

The term was first introduced by a general surgeon named George Hackett in 1956, who proposed that injecting sclerosing solutions could improve joint stability by tightening interarticular ligaments and joint capsules [1] [2] . The word “prolotherapy” consists of two following root words: The Latin “proles,” which means proliferation and offspring, and the English word “therapy” [1] . Dr. George Hackett initially formalized Prolotherapy injection protocols, discussing injecting small amounts of a solution into the affected site during several treatment sessions [4] .

Prolotherapy has been considered an alternative treatment for a wide variety of painful musculoskeletal conditions that are usually refractory to standard therapies [4] . Protocol of injection varied from one condition to another, depending on several variables such as damage type and location, clinical severity, and practitioner preference [4] [5] . Prolotherapy is performed based on palpating tender points in affected sites [6] [7] , using anatomic landmarks [8] or under ultrasound or fluoroscopic guidance [5] [9] . There are multiple solutions used in prolotherapy, categorized into three major groups. Some agents are considered irritants (including phenol or Phenol-glycerin-glucose, guaiacol, and tannic acid), some are chemoattractants (sodium morrhuate), and others are known as osmotic agents (including hypertonic glucose, glycerin, and zinc sulphate) [10] . Even a mixture of these solutions can be used in treating musculoskeletal problems [7] . Chemoattractants act as vascular sclerosants and attract inflammatory mediators to the injury site [11] . Sodium morrhuate is the one and only member of this group [10] . Osmotic solutions create a hypertonic environment, leading to dehydration, cell rupture and osmotic shock, which in turn result in granulocytes and macrophages attraction and induce collagen deposition which ultimately stimulates tissue healing process. They also upregulate the expression of platelet-derived growth factor in injured areas [2] . Hypertonic dextrose (D-glucose) has been categorized as an osmotic agent. It is the most commonly used solution in practice that is water-soluble and considered one of the normal constituents of blood chemistry [1] . The third class, Irritants, directly attack cells and kill them by presenting their antigens at cellular surface through changing cell surface proteins [10] . In many of the earlier published studies, phenol-glycerin-glucose was included as an irritant substance, but it is no longer used today [11] . In vitro studies indicated that human fibroblasts and chondrocytes exposure to only 0.5% concentrations of dextrose have stimulated the production of several growth factors, which are essential to the functional and structural repair of tendinous and ligamentous tissues. These substances include platelet-derived growth factor, transforming growth factor β, epidermal growth factor, basic fibroblast growth factor, insulin-like growth factor, and connective tissue growth factor. In vitro studies demonstrate that the mentioned growth factors have promoted the expression of types 1 and 3 collagens which pertain to the growth of tendon, ligament, and cartilage. Growth factors production and cellular proliferation are the critical factors of tissue repair in prolotherapy injection. While using the concentrations of 0.5% dextrose prolotherapy showed promising results in experimental studies, dextrose concentrations higher than 10% are considered as inflammatory concentrations in clinics and be utilized as irritant agents in prolotherapy. Concentrations less than 10% are non-inflammatory [2] . Although the exact mechanism of action remains unclear, it is hypothesized that prolotherapy solutions may induce a local inflammatory response at the site of injection, which leads to fibroblast proliferation and subsequent collagen synthesis, resulting in stronger and tighter ligaments and tendons, reduction in pain and dysfunction and also improved joint stability and biomechanics [4] [5] .

The effect of prolotherapy in pain reduction is unclear, but investigations reported that dextrose could alleviate pain through blocking transient receptor potential vaniloid 1 (TRPV1) pain receptors and even modulate sensorineural pain receptors [1] .

In vitro studies demonstrated that in the environment of highly concentrated glucose, the expression of collagen type 1 and 3 genes are increased in fibroblasts, and the matrix protein aggrecan amounts is changed in chondrocytes, which could contribute to tissue regeneration in peri-articular structures. There is an increase in cellularity and fibrous components in the cartilage tissue in the hyperosmolar conditions [1] .

This review has especially focused on dextrose prolotherapy protocols, as it is the most common prolotherapy substance in practice [1] .

Rotator cuff tendinopathy is chronic overuse tendinopathy that is determined by pain and weakness during shoulder movements of external rotation and elevation Shoulder pain is categorized as the third common cause of musculoskeletal pain, and tendinopathy of rotator cuff is the most common generator of that pain [8] . There is no consensus on the optimal treatment utilized for rotator cuff tendinopathy. Many typical treatments have been proposed, including exercise therapies, oral anti-inflammatory drugs, interventional therapies, and surgical procedures [8] [33] . Exercise therapy is generally counted as the first treatment choice in patients with impingement symptoms [34] . However, these exercises are often supplemented by adjunct therapies such as injection of corticosteroids, platelet-rich plasma, prolotherapy solutions, and hyaluronic acid. Corticosteroid is the most common substance in injections, but its long-term application is not recommended regarding to the deleterious effects that it can has on tendon integrity and further surgical outcomes [34] . Corticosteroid adjuvant therapy can be replaced by other injection therapies, such as prolotherapy. During the past decades, there has been emerging evidence in the effectiveness of prolotherapy for treating rotator cuff tendinopathy. They include various injection protocols and techniques to strengthen rotator cuff tendons, improve shoulder function and reduce pain. Initially, a landmark-based approach was performed for injecting prolotherapy solution; however, as we move forward, more accurate injections have been performed using ultrasound as a guide [8] . Dextrose injection in Rotator cuff tendinous structures results in a short-term improvement of pain, functional status, and ROM and is associated with compelling long-term follow-up results [8] . In long-term evaluation, prolotherapy has more significant effects than placebo injection on pain reduction [34] . Although, investigations suggest prolotherapy as an effective treatment, especially when performed through multiple-site injection techniques and in higher concentrations [33] . Compared to arthroscopic surgery techniques, the rate of complications is lower in prolotherapy. The variable results in prolotherapy studies may be related to the heterogeneity of treatment protocols varying in dextrose concentration, volume of injection, number of injection sites and injection intervals for rotator cuff tendinopathy [8] [33] [34] [35] .

Lateral epicondylitis (tennis elbow) is a painful enthesopathy at the common extensor tendon in the elbow [36] . The disorder usually develops due to repetitive and intensive use of hand extensor muscles [37] . It is commonly observed in industrial workers and tennis players [38] . The condition occurs when normal tendon repair process fails to be completed, which leads to tendon degenerative changes and micro tears. The effect of local inflammation in pathology is slight [36] . Although most cases are self-limited and resolved between 6 to 12 months, 20% of cases became chronic and resistant to usual care [38] . Treatment options include operative and non-operative therapies. Physiotherapy and exercise-based rehabilitation are the primary choices for treatment. Second-line interventions include corticosteroid injection, shock wave therapy, braces and analgesics, and biological treatments [38] . Prolotherapy is a biological therapy that has shown promising results in pain improvement and functional assessment in terms of lateral epicondylitis treatment. Prolotherapy is widely compared with saline injection as a control group. Injection for the control groups is performed using the same technique of prolotherapy injection. It is hypothesized that the control injection can have a therapeutic effect by triggering cellular rupture and local bleeding [39] . There is no consensus on the number of treatment sessions, frequencies, concentrations, and dosages of the solution, and the injection techniques. The prolotherapy solution comprises hypertonic dextrose alone or in a mixture with other irritant substances, including sodium morrhuate, glycerin, or phenol. The concentration ranges from 10% to 25%, with 20% as the most frequent concentration. Treatment sessions widely vary in number and frequency. In most cases, one or more therapeutic sessions, with a mode of three, are accomplished for each patient every three or four weeks. In some studies, practitioners used peppering techniques for injection, while in other studies, they didn’t. The injection can be appliedto the points of tenderness around the elbow or, more commonly, into the epicondyle region and adjacent areas such as supracondylar ridge, annular and radial collateral ligament, and insertion of the extensor carpal muscles [39] [40] . Frequently measured outcomes include pain (VAS score), functional status of the elbow measured through disabilities of the arm, shoulder and hand (DASH)and The Patient-Rated Tennis Elbow Evaluation (PRTEE) questionairres, and handgrip strength. Different studies indicated that prolotherapy injection could significantly improve all of the mentioned outcomes [32] [37] [39] . Some studies reported that in comparison with other interventions, prolotherapy had significant effects on pain reduction and functional improvement in medium and long-term follow-up, while in the short-term, it doesn’t accompany substantial results [40] .

Achilles tendinopathy is the most frequent Achilles-related disorder in athletes and the general population [41] . The condition involves increased tendon thickness, decreased vascularity, and chronic tendinitis. Patients present with local pain and swelling over a specific area of a thickened tendon. Foot range of motion may be affected by decreased plantar flexion. Mid-portion Achilles tendinopathy and insertional Achilles tendinopathy are two major types of the disorder [42] . Various therapeutic modalities for Achilles tendinopathy include immobilization with night splints, physical therapy and eccentric loading exercises, shockwave therapy, nitrate oxide, cryotherapy, physiotherapy, and injection therapy [41] . Eccentric loading exercise (ELE) is considered a standard conservative treatment with promising results [6] . Prolotherapy can be used alone or as an additive to ELE treatment. The injection is performed at each tender point, usually located in the anterolateral and anteromedial border of the tendon and on the most posterior aspect of the tendon 2 - 7 cm away from the insertion point [41] . Treatment duration will be until the pain relief is achieved. Studies demonstrated that prolotherapy injection has more significant effects than eccentric loading exercise on short-, intermediate-, and long-term pain. It is also indicated that a combination of prolotherapy and ELE resulted in earlier stiffness reduction and activity improvement [41] . Some studies denied the beneficial therapeutic effect of prolotherapy alone when that doesn’t accompany eccentric loading exercises. Improvement in Victorian Institute of Sports Assessment—Achilles score (VISA-A) score increased vascularity, and reduction in tendon thickening can be expected as a result of prolotherapy injections in Achilles tendinopathy [43] .

Plantar fasciitis is one of the most common causes of heel pain, associated with a high burden of disability [44] . The condition affects approximately 10% of the population and is more prevalent among runners and middle-aged people [45] . Clinical manifestation involves pain alongside the calcaneal enthesis early in the morning and during the first steps after prolonged standing or sitting [46] . Ethiopatology is characterized by collagen degeneration at the fascia attachment point due to micro tears resulting from overuse tendinopathy [47] . Diagnosis of plantar fasciitis is based on history and clinical evaluation, although ultrasonography can be used as a further diagnostic instrument in challenging cases. Plantar fascia thickness greater than 4 mm and the presence of hypoechogenic areas are sonographic features supporting the diagnosis of plantar fasciitis [48] . Common conservative therapies, including nonsteroidal anti-inflammatory drugs, stretching exercises, arch supports, dorsiflexion night splints, extracorporeal shockwave therapy (ESWT), physical therapy, and injection therapies considered first-line treatment [48] . The recovery rate is 70% - 80% in conventional therapies but nearly 10% of patients remain recalcitrant to treatment [44] [49] . Recent complementary treatment options such as ESWT and injection therapies showed compelling results in treating chronic and resistant cases. Prolotherapy is administered to improve the patient’s condition. Prolotherapy could be performed in one or more sessions, frequently in three sessions. Patients receive prolotherapy agents in a regular weekly order, usually every three weeks [46] [47] [48] [50] [51] . Studies reported that repeated injections are associated with the most accurate and effective results [51] . A concentration of dextrose 15% or 20% is frequently used [48] [52] . Injection is performed under palpation guidance or ultrasound guidance. It is recommended to use the ultrasound method, regarding its functionally better outcomes. Although studies determine that the palpation method is associated with a higher rate of disease recurrence [51] . In the ultrasound method, an ultrasound probe is placed over the fascia to reveal the thickness and echogenicity of different parts. A needle is inserted medially, perpendicular to the long axis of the transducer, approaching the most thickened and hypoechogenic area under sonographicguidance [49] [51] . In the palpation method, the injection can be administered in various techniques, including needle insertion into the most tender point of the heel [47] or injection at five different points of the fascia, including the attachment point of plantar fascia at heel (medial and lateral), at metatarsal bones (top of the first and fifth metatarsus), and at the middle part of the fascia [48] Peppering technique is considered in many injections [48] [49] . Corticosteroid injection, normal saline injection, platelet-rich plasma (PRP) injection, ESWT, and stretching exercises are common control groups used to compare prolotherapy effects with other procedures. Measured outcomes are categorized into three different classes, including pain assessment during rest and activity through VAS score, and NRS (numeric rating scale), functional assessment (foot functional index (FFI), foot and ankle ability measure (FAAM), The American Orthopedic Foot and Ankle Score (AOFAS), and sonographic features (plantar fascia thickness, echogenicity). Studies indicate that dextrose prolotherapy is associated with significant improvement in pain (at rest and activity), FFI and FAAM scores, and plantar fascia thickness and hypoechogenicity in patients with plantar fasciitis [47] [48] . Compared with non-active treatments (normal saline injection and exercises), prolotherapy is significantly better at improving short-term pain [53] . Prolotherapy has been more effective than corticosteroid injection in long-term outcomes, while corticosteroid injection demonstrated more significant improvements in short-term evaluation [44] . Results didn’t significantly differ between ESWT groups and prolotherapy [52] . PRP injection is more effective than dextrose prolotherapy in FFI score improvement [49] .

Osgood-Schlatter disease (OSD) is a common cause of chronic knee pain associated with disabilities in daily work and sport participation [54] . Nonsteroidal anti-inflammatory drugs, knee paddling, and physical and surgical therapies are usual conservative treatments for OSD. Most patients respond to conventional therapies; however, some remain resistant [55] . Only a few studies aimed to assess the effectiveness of prolotherapy on Osgood-Schlatter, making it difficult to reach a reliable conclusion. Injections performed under sonographic guidance or based on anatomical landmarks. Deep infrapatellar bursa, infrapatellar fat pad, and superficial infrapatellar bursa are frequent injection sites when ultrasound guidance is applied [55] . Monthly injections in a total period of three months are a usual method [55] [56] . Hyperosmolar dextrose prolotherapy can result in a rapid asymptomatic sport participation, greater pain reduction during activity, and significant improvement in functional status which is measured by Victorian Institute of Sports Assessment—Patellar Tendon (VISA-P) score [2] [54] [55] [56] .